Mutant endonuclease V enzymes and applications thereof

ABSTRACT

Provided herein are mutant endonuclease V enzymes that are capable of nicking an inosine-containing DNA sequence. Nucleic acid assays and agents that employ such mutant endonuclease V enzymes to introduce a nick into a target DNA including one or more inosine, and uses a DNA polymerase to generate amplicons of a target DNA are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of continuation-in-partU.S. patent application Ser. No. 13/840,062, filed on Mar. 15, 2013,entitled “MUTANT ENDONUCLEASE V ENZYMES AND APPLICATIONS THEREOF”, whichis a continuation-in-part of U.S. patent application Ser. No.13/330,745, filed on Dec. 20, 2011, entitled “ISOTHERMAL DNAAMPLIFICATION”, which is a divisional of U.S. patent application Ser.No. 11/621,703, filed on Jan. 10, 2007, now U.S. Pat. No. 8,202,972 B2,entitled “ISOTHERMAL DNA AMPLIFICATION”.

FIELD OF INVENTION

The present invention generally relates to endonuclease V mutant enzymesthat are capable of introducing a nick in a DNA sequence comprising aninosine residue that is base-paired with a cytosine residue and theiruse in nucleic acid assays that include nicking of DNA sequencescontaining inosine nucleotides. It further relates to improved DNAamplification methods wherein a nick is introduced in a double stranded,target DNA by an endonuclease V mutant followed by amplification of thetarget DNA. Kits comprising such engineered endonuclease V are alsodisclosed.

BACKGROUND

DNA amplification is a process of copying a single or double-strandedtarget DNA to generate multiple copies of the target DNA. Since DNAstrands are antiparallel and complementary, each strand may serve as atemplate (template strand) for the production of an opposite strand(complementary strand) by a DNA polymerase. The template strand ispreserved as a whole or as a truncated portion and the complementarystrand is assembled from nucleoside triphosphates. A variety oftechniques are currently available for efficient amplification ofnucleic acids such as polymerase chain reaction (PCR), ligase chainreaction (LCR), self-sustained sequence replication (3SR), nucleic acidsequence based amplification (NASBA), strand displacement amplification(SDA), multiple displacement amplification (MDA), or rolling circleamplification (RCA). Many of these techniques generate a large number ofamplified products in a short span of time. For example, in a polymerasechain reaction (PCR), a target DNA, a pair of primers and a DNApolymerase are combined and subjected to repeated temperature changesthat permit melting, annealing, and elongation steps to result in anexponential amplification of the starting target DNA. However, in PCR,the melting or denaturation step typically occurs at a high temperaturelimiting the choice of polymerases to thermophilic polymerases.

DNA amplification often suffers from high background signals, which aregenerated by non-specific amplification reactions yieldingundesired/false amplification products. For example, non-specificamplification may result from various primer gymnastics such as nucleicacid template-independent primer-primer interactions. Primers may formprimer-dimer structures by intra- or inter-strand primer annealing(intra molecular or inter molecular hybridizations), and may getamplified, and may sometimes predominate, inhibit, or mask theamplification of a target DNA sequence. Such non-specific, backgroundamplification reactions become even more problematic where the targetnucleic acid to be amplified is available only in limited quantities(e.g., whole-genome amplification from a single DNA molecule). EfficientDNA amplification techniques are needed, if they are to be used forcritical applications such as diagnostic applications, wherein afalse-positive amplification may likely result in a wrong diagnosis.

Endonuclease V (also referred as endo V or deoxyinosine 3′ endonuclease)is a DNA repair enzyme that recognizes DNA containing deoxyinosine (adeamination product of deoxyadenosine, also referred as inosine)residues. Endonuclease V primarily cleaves the second or thirdphosphodiester bond 3′ to an inosine residue in the same strand leavinga nick with a 3′-hydroxyl and a 5′-phosphate. Endonuclease V was firstdescribed in Escherichia coli (E. coli). Apart from inosine residues, E.coli endonuclease V also recognizes, to a lesser degree, otherwisemodified bases such as abasic sites (AP sites) or urea, base mismatches,insertion/deletion mismatches, hairpin or unpaired loops, flaps andpseudo-Y structures. One or more embodiments of the present inventionare directed towards engineered endonuclease V enzymes (mutantendonuclease V) and their use in nucleic acid assays such as stranddisplacement DNA amplification reactions, wherein the selective nickingcapability of mutant endonuclease V is employed to develop an improvedmethod of DNA amplification.

BRIEF DESCRIPTION

One or more embodiments of the present invention are directed tomethods, agents, and kits for producing amplification products (i.e.,amplicons) from a target DNA using an endonuclease V and aninosine-containing primer. In some embodiments, the methods comprise thesteps of (a) providing a target DNA; (b) annealing at least oneinosine-containing primer to the target DNA to create a targetDNA:primer hybrid; (c) nicking the inosine-containing primer in thetarget DNA:primer hybrid with an endonuclease at a residue 3′ to theinosine residue; and (d) extending the nicked inosine-containing primervia a nucleic acid amplification to produce at least one ampliconcomplementary to at least one portion of the target DNA.

In some embodiments, a method of producing at least one amplicon basedon a target DNA is provided, wherein the method includes (a) providing atarget DNA; (b) annealing at least one inosine-containing primer to thetarget DNA to produce a target DNA:primer hybrid; (c) extending theprimer strand via a nucleic acid amplification reaction to produce acomplementary strand to at least one portion of the target DNA and togenerate a nicking site in the extended primer strand at a residue 3′ tothe inosine residue; (d) nicking the extended primer strand at thenicking site to generate an initiation site in the primer strand for asubsequent nucleic acid amplification reaction; and (e) repeating steps(c) and (d) employing a strand displacement nucleic acid polymerase forthe nucleic acid amplification reaction to produce the at least oneamplicon based on the target DNA.

In some embodiments, a method of producing at least one amplicon basedon a target DNA is provided, wherein the method comprises: (a) providingthe target DNA; (b) providing a DNA amplification reaction mixturecomprising at least one inosine-containing primer, at least one 5′→3′exonuclease-deficient DNA polymerase having strand displacementactivity, at least one nuclease that is capable of nicking a DNA at aresidue 3′ to an inosine residue, and dNTP mixture; and (c) amplifyingat least one portion of the target DNA using the DNA amplificationreaction mixture of step (b) to produce the at least one amplicon.

In some embodiments, amplicon production kits are provided thatcomprises at least one inosine-containing primer, at least oneexonuclease-deficient DNA polymerase with strand displacement activityand at least one nuclease that is capable of nicking DNA at a residue 3′to an inosine residue.

In some embodiments, mutant endonuclease V enzymes are provided. In someembodiments, the amino acid sequence of the mutant endonuclease Vcomprises a modified sequence of SEQ ID NO: 1, wherein the modificationis a replacement of a Tyrosine residue at the 75^(th) position of theSEQ ID NO: 1 with an Alanine residue. In some embodiments, a mutant E.coli endonulcase V comprising the amino acid sequence of SEQ ID NO: 2,or its conservative variants are provided. In some other embodiments, amutant Archaeoglobus fulgidus (A. fulgidus or Afu), comprising the aminoacid sequence of SEQ ID NO: 3, or its conservative variants areprovided. The amino acid sequence of the mutant Archaeoglobus fulgidusendonuclease V is a modified sequence of SEQ ID NO: 56, wherein themodification is a replacement of a Tyrosine residue at the 74^(th)position of the SEQ ID NO: 56 with an Alanine residue.

In some embodiments, a nucleic acid assay is disclosed that includesproviding a target DNA, a DNA polymerase and a mutant endonuclease Vthat is capable of nicking an inosine-containing strand of a doublestranded DNA at a residue 3′ to the inosine residue when the inosineresidue is base-paired with a cytosine residue. A double stranded DNA isthen generated from the target DNA, wherein the double stranded DNAcomprises an inosine residue base-paired with a cytosine residue. Theinosine-containing strand of the double stranded is subsequently nickedemploying the mutant endonuclease V to generate a nicked DNA. Thenucleic assay then includes conducting a DNA polymerase reaction on thenicked DNA employing the DNA polymerase.

DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying figures.

FIG. 1 shows a general scheme for inosine-based amplicon production.

FIG. 2 depicts several nucleic acid synthesis schemes that employ anendonuclease V presented as a single series. The synthesis schemes shownin FIG. 2 provide methods for generating plus strands from a target DNAusing a forward primer (hereinafter referred as “Ping” product);generating minus strands using a reverse primer (hereinafter referred as“Pong” product); addition of a promoter to the DNA product using anextender template; and generating RNA products using an RNA polymerasecapable of initiating synthesis at the promoter that was added with theextender template.

FIG. 3 shows a general scheme for detecting amplicons using pairedfluorescent and quenching chromophores attached to oligonucleotidesconnected by hybridization to an extender template.

FIG. 4 depicts the use of a variety of polymerases to produce ampliconsfrom a target DNA (“Ban 1”), in which a single primer (expected productof 61 nucleotides) or multiple primers (forward and reverse, withexpected products of 61 nucleotides (61′-mer) and 45 nucleotides(45′-mer)) were employed as described in the Example 2.

FIG. 5 depicts amplicon production using multiple sets of nested primersin several different reaction mixtures as described in Example 4.

FIG. 6A and FIG. 6B depict in vitro transcription, variations indivalent cations, and variations in single strand binding proteinsdescribed in Example 5. FIG. 6A shows a gel with the DNA products andFIG. 6B shows a gel with the RNA products.

FIG. 7 shows the reaction products using a variety of SSB concentrationsas described in Example 6.

FIG. 8 depicts amplicon extension using an extension template asdescribed in Example 7.

FIG. 9 depicts amplification using genomic DNA template as described inExample 8.

FIG. 10 shows amplicon generation from lambda DNA (102 nucleotideproducts) and the effects of contaminating DNA as described in Example9.

FIG. 11A and FIG. 11B depict the relative activities of the wild-type(WT) endonuclease V to the activity of the mutant endonuclease V asdescribed in Example 12.

FIG. 12 demonstrates that both WT and mutant endonuclease V nucleasesact on inosine-containing DNA but substantially not on theguanine-containing DNA as described in Example 13.

FIG. 13 demonstrates that the nuclease/polymerase combination (Y75Amutant E. coli endonuclease V and exo 9-) Bst polymerase) generatesamplicon DNA from inosine-containing DNA but not on theguanine-containing DNA as described in the Example 14.

FIG. 14 depicts the results of a series of experiments that demonstratethe ability of the Y74A mutant A. fulgidus (Afu) endonuclease V (SEQ IDNO: 3, wherein the Tyrosine residue at the 74^(th) position of WT Afuendo V is replaced with an Alanine residue) and the Y75A mutant E. coliendonuclease V (SEQ ID NO: 2, wherein a Tyrosine residue at the 75^(th)position of WT E. coli endo V (SEQ ID No: 1) is replaced with an Alanineresidue) to function with polymerase to generate amplicon from a targetDNA as described in Example 15.

FIG. 15 shows the thermal stability of the Y75A mutant E. coliendonuclease V (SEQ ID NO:2) at a variety of temperatures as describedin Example 16.

FIG. 16 shows the results of real-time DNA amplification as described inExample 17.

FIG. 17 shows the Y80A mutant Tma endonuclease V (SEQ ID NO: 58)activity on single stranded DNA and double stranded DNA as described inExample 18.

FIG. 18 shows the nicking efficiency of Tma endonuclease V on doublestranded DNA and single stranded DNA at 45 C and 60 C as described inExample 19.

FIG. 19 shows the nicking efficiency of Tma endonuclease V on doublestranded DNA at 60° C. as described in Example 20.

FIG. 20 shows the nicking efficiencies of Y75A mutant E. coliendonuclease V (SEQ ID NO: 2) and Y80A mutant Termotoga maritima (Tma)endonuclease V (SEQ ID NO: 58) as described in Example 21.

FIG. 21 shows the nicking efficiencies of Y75A mutant E. coliendonuclease V (SEQ ID NO: 2) on single stranded DNA and double strandedDNA in various buffers as described in Example 22.

DETAILED DESCRIPTION

The following detailed description is exemplary and not intended tolimit the invention of the application and uses of the invention.Throughout the specification, exemplification of specific terms shouldbe considered as non-limiting examples. The singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Approximating language, as used herein throughout thespecification and claims, may be applied to modify any quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. Unless otherwise indicated, all numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Where necessary,ranges have been supplied, and those ranges are inclusive of allsub-ranges there between. To more clearly and concisely describe andpoint out the subject matter of the claimed invention, the followingdefinitions are provided for specific terms, which are used in thefollowing description and the appended claims.

As used herein, the term “biological sample” refers to a sample obtainedfrom a biological subject. It includes samples obtained in vivo or invitro. Biological sample includes, but are not limited to, body fluid(e.g., blood, blood plasma, serum, or urine), organs, tissues, fractionsand sections (e.g., sectional portions of an organ or tissue) and cellsisolated from a biological subject or from a particular region (e.g., aregion containing diseased cells) of a biological subject. Thebiological sample contains or is suspected to contain a target nucleicacid. The biological sample may be of eukaryotic origin, prokaryoticorigin, viral origin or bacteriophage origin. Biological sample may beobtained from an insect, a protozoa, a bird, a fish, a reptile, a mammal(e.g., rat, mouse, cow, dog, guinea pig, or rabbit), or a primate (e.g.,chimpanzee or human). Biological samples may be dispersed in solution ormay be immobilized on a solid support, such as in blots, assays, arrays,glass slides, microtiter, or ELISA plates.

As used herein, the term “target DNA” refers to a DNA sequence ofnatural or synthetic origin that may be synthesized or amplified usingone of more of the methods of the present invention. The term “targetDNA template” refers to a portion of the target DNA that may be used bya DNA polymerase to produce one or more amplicons.

As used herein, the term “complementary”, when used to describe a firstnucleic acid/oligonucleotide sequence in relation to a second nucleicacid/oligonucleotide sequence, refers to the ability of a polynucleotideor oligonucleotide comprising the first nucleic acid/oligonucleotidesequence to hybridize (e.g., to form a duplex structure) under certainhybridization conditions with an oligonucleotide or polynucleotidecomprising the second nucleic acid/oligonucleotide sequence.Hybridization occurs via base pairing of nucleotides (complementarynucleotides). Base pairing of the nucleotides may occur via Watson-Crickbase pairing, non-Watson-Crick base pairing or base pairing formed bynon-natural/modified nucleotides. For example, an adenine (A) base in anadenosine is capable of base paring with a thymine (T) base in athymidine; and a guanine (G) base in a guanosine is capable of basepairing with a cytosine (C) base in a cytidine via Watson-Crick hydrogenbonding. Thus A is considered to be complementary to T, and G isconsidered to be complimentary to C. If a nucleotide at a certainposition of a first oligonucleotide strand is capable of hydrogenbonding with a nucleotide at a corresponding position of a secondoligonucleotide strand, then the two oligonucleotide strands areconsidered to be complementary to each other at that position. Twooligonucleotide strands are considered complementary to each other as awhole when a sufficient number of corresponding positions in each of thetwo oligonucleotides have nucleotides that hydrogen bond with eachother.

As used herein, the term “dNTP mixture” refers to a mixture ofdeoxynucleotide triphosphates that act as precursors required by a DNApolymerase for DNA synthesis. Each of the deoxynucleotide triphosphatesin a dNTP mixture comprises a deoxyribose sugar, an organic base, and aphosphate in a triphosphate form. A dNTP mixture may include each of thenaturally occurring deoxynucleotide triphosphate (e.g., dATP, dTTP,dGTP, dCTP or dUTP). In some embodiments, each of the naturallyoccurring deoxynucleotide triphosphates may be replaced or supplementedwith a synthetic analog; provided however that inosine base may notreplace or supplement guanosine base (G) in a dNTP mixture. Each ofdeoxynucleotide triphosphate in dNTP may be present in the reactionmixture at a final concentration of 10 μM to 20,000 μM, 100 μM to 1000μM, or 200 μM to 300 μM.

As used herein, the term “inosine” or “inosine residue” refers to a2′-deoxyribonucleoside or 2′-ribonucleoside residue wherein thenucleobase is a hypoxanthine. Xanthine structures are alternatestructures to inosine residues, resulting from deamination of guanine.The inosine residue is capable of base pairing with a thymine, acytidine or a uridine residue. The term “inosine analog” refers to a2′-deoxyribonucleoside or 2′-ribonucleoside wherein the nucleobaseincludes a modified base such as xanthine, uridine, oxanine (oxanosine),other O-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deazahypoxanthine, other 7-deazapurines, and 2-methyl purines. The term“inosine-containing primer” refers to a primer sequence including atleast one inosine residue. In some embodiments, the inosine-containingprimer may comprise an inosine analog, including xanthine. A nucleicacid sequence containing inosine or inosine analogue residue as referredherein are recognized and cleaved by an endonuclease V.

As used herein, the term “amplicon” refers to nucleic acid amplificationproducts that result from the amplification of a target nucleic acid.Amplicons may comprise a mixture of amplification products (i.e., amixed amplicon population), several dominant species of amplificationproducts (i.e., multiple, discrete amplicons), or a single dominantspecies of amplification product. A single species of amplicon may beisolated from a mixed population of amplicons using art-recognizedtechniques, such as affinity purification or electrophoresis. Anamplicon may comprise single-stranded or double-stranded DNA, DNA:RNAhybrids, or RNA depending on the reaction scheme used. An amplicon maybe largely single-stranded or partially double-stranded or completelydouble-stranded DNA, DNA:RNA hybrids, or RNA.

As used herein, the term “primer”, or “primer sequence” refers to ashort linear oligonucleotide that hybridizes to a target nucleic acidsequence (e.g., a target DNA template to be amplified) to prime anucleic acid synthesis reaction. The primer may be a RNAoligonucleotide, a DNA oligonucleotide, or a chimeric sequence. Theprimer may contain natural, synthetic, or modified nucleotides. Forexample, the primer may comprise naturally occurring nucleotides (G, A,C or T nucleotides) or their analogues. Suitable primers may alsoinclude at least one inosine residue (e.g., inosine-containing primer)positioned near the 3′ terminal end of the primer (e.g., as penultimatenucleotide at the 3′ end of a primer sequence). Both the upper and lowerlimits of the length of the primer are empirically determined. The lowerlimit on primer length is the minimum length that is required to form astable duplex upon hybridization with the target nucleic acid undernucleic acid amplification reaction conditions. Very short primers(usually less than 3 nucleotides long) do not form thermodynamicallystable duplexes with target nucleic acid under such hybridizationconditions. The upper limit is often determined by the possibility ofhaving a duplex formation in a region other than the pre-determinednucleic acid sequence in the target nucleic acid. Generally, suitableprimer lengths are in the range of about 3 nucleotides long to about 40nucleotides long. In some embodiments the primer ranges in length from 5nucleotides to 25 nucleotides. As used herein the term “forward primer”refers to a primer that anneals to a first strand of the target DNA andthe term “reverse primer” refers to a primer that anneals to acomplimentary, second strand of the target DNA. Together a forwardprimer and a reverse primer are generally oriented on the target DNAsequence in a manner analogous to PCR primers, such that a DNApolymerase can initiate the DNA synthesis resulting in replication ofboth the strands.

As used herein, the term “melting temperature” of a primer refers to thetemperature at which 50% of primer in a primer-DNA hybrid dissociatesinto free primer and DNA. The melting temperature of a primer increaseswith its length. The melting temperature of a primer also depends on itsnucleotide composition. Thus primers with many G and C nucleotides willmelt at a higher temperature than ones that only have A and Tnucleotides. High melting temperatures (e.g., above 65° C.) and veryhigh melting temperatures (e.g., above 80° C.), may be disfavoredbecause some DNA polymerases denature and lose activity at such hightemperatures. Because ionic strength also affects the meltingtemperature of a primer, all melting temperature values provided hereinare determined at a pH of 7.7 with 5 mM MgCl₂ and 50 mM NaCl.

As used herein, the term “reducing agent” refers to agent that hascapability to reduce disulfides to mercaptans. Suitable reducing agentsmay contain thiol groups such as dithiothreitol (DTT), 2-mercaptoethanol(βME), and 2-mercaptoethylamine (MEA). Alternatively, reducing agentsmay contain phosphines and their derivatives, for example,Tris(carboxyethyl) phosphine (TCEP).

As used herein, the term “single strand DNA binding protein”,abbreviated as “SSB”, refers to proteins that bind non-covalently tosingle stranded DNA with a higher affinity than to double stranded DNA.Suitable examples of single strand binding proteins include, but are notlimited to, E. coli SSB, T4 gene 32 protein, T7 gene 2.5 protein, Ncp7,recA, or combinations thereof.

As used herein, the term “vector” refers to any autonomously replicatingor integrating agent, including but not limited to, plasmids, cosmids,and viruses (including phage). The vector comprises a nucleic acidsequence to which one or more additional nucleic acid sequence ofinterest may be included. The vector may be an expression vector bywhich the amino acid sequences corresponding to the nucleic acidsequences of interest may be expressed in a suitable host. Vectors maybe used both to amplify and to express DNA (e.g., genomic or cDNA) orRNA.

As used herein, the term “transformed cell” refers a cell into which (ora predecessor or an ancestor of which) a nucleic acid sequence encodinga polypeptide of interest has been introduced, by means of, for example,recombinant DNA techniques or viruses.

A “purified” or “isolated” polypeptide or polynucleotide is one that issubstantially free of the materials with which it is generallyassociated in nature. By substantially free is meant at least 50%,preferably at least 70%, more preferably at least 80%, even morepreferably at least 90% free of the materials with which it isassociated in nature.

The term “conservative variants”, as used herein, applies to both aminoacid and nucleic acid sequences. With respect to particular nucleic acidsequences, the term “conservative variants” refers to those nucleicacids that encode identical or similar amino acid sequences (i.e., aminoacid sequences that have similar physico-chemical properties) andinclude degenerate sequences. For example, the codons GCA, GCC, GCG, andGCU all encode alanine. Thus, at every amino acid position where analanine is specified, any of these codons may be used interchangeably inconstructing a corresponding nucleotide sequence. Such nucleic acidvariants are conservative variants, since they encode the same protein(assuming that is the only alternation in the sequence). One skilled inthe art recognizes that each codon in a nucleic acid, except for AUG(sole codon for methionine) and UGG (tryptophan), may be modifiedconservatively to yield a functionally identical peptide or proteinmolecule. As to amino acid sequences, one skilled in the art willrecognize that alteration of a polypeptide or protein sequence viasubstitutions, deletions, or additions of a single amino acid or a smallnumber (typically less than about ten) of amino acids may be a“conservative variant” if the physico-chemical properties of the alteredpolypeptide or protein sequence is similar to the original. In somecases, the alteration may be a substitution of one amino acid with achemically similar amino acid. Examples of conservative variantsinclude, but not limited to, the substitution of one hydrophobic residue(e.g., isoleucine, valine, leucine or methionine) for one another; orthe substitution of one polar residue for another (e.g., thesubstitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine) and the like. Genetically encoded amino acidsgenerally may be divided into four families: (1) acidic: aspartate,glutamate; (2) basic: lysine, arginine, histidine; (3) nonpolar:alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar: glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine.

The term “mutant endonuclease” or “engineered endonuclease” as usedherein refers to an endonuclease enzyme that is generated by geneticengineering or protein engineering, wherein one more amino acid residuesare altered from the wild type endonuclease. The alteration may includea substitution, a deletion or an insertion one or more amino acidresidues. Throughout the specification and claims, the substitution ofan amino acid at one particular location in the protein sequence isreferred using a notation “(amino acid residue in wild typeenzyme)(location of the amino acid in wild type enzyme)(amino acidresidue in engineered enzyme)”. For example, a notation Y75A refers to asubstitution of a Tyrosine (Y) residue at the 75^(th) position of thewild type enzyme by an Alanine (A) residue (in mutant/engineered enzyme)

One or more embodiments of the present invention provide compositions,methods and kits for various nucleic acid assays, wherein anendonuclease that is capable of nicking an inosine-containing strand ofa double-stranded nucleic acid at a location 3′ to the inosine residueis employed. In some embodiments, the endonuclease is a geneticallyengineered endonuclease.

For nucleic acid assays, samples suspected or known to contain aparticular target nucleic acid sequence may be obtained from a varietyof sources. The sample may be, for example, a biological sample, a food,an agricultural sample, or an environmental sample. Samples may also bederived from a variety of biological subjects. The biological subjectmay be of prokaryotic or eukaryotic origin and includes viruses. Thesample may be derived from a biological tissue or a body fluid or anexudate (e.g., blood, plasma, serum or urine, milk, cerebrospinal fluid,pleural fluid, lymph, tears, sputum, saliva, stool, lung aspirates,throat or genital swabs, and the like), whole cells, cell fractions, orcultures.

The target nucleic acid for a nucleic acid assay may be dispersed insolution or immobilized on a solid support (such as blots, arrays,microtiter, or well plates). A sample may be pretreated to make thetarget nucleic acid available for hybridization. For example, when atarget nucleic acid is in a double stranded form, it may be denatured togenerate a single stranded form of the target DNA. The target doublestranded DNA may be thermally denatured, chemically denatured, or boththermally and chemically denatured. In some embodiments, the doublestranded DNA is chemically denatured using a denaturant (e.g., glycerol,ethylene glycol, formamide, or a combination thereof) that reduces themelting temperature of double stranded DNA. In certain embodiments, thedenaturant reduces the melting temperature by 5° C. to 6° C. for every10% (vol./vol.) of the denaturant added to the reaction mixture. Thedenaturant or combination of denaturants may comprise 1%, 5%, 10%(vol./vol.), 15% (vol./vol.), 20% (vol./vol.), or 25% (vol./vol.) ofreaction mixture. In certain embodiments, the denaturant comprisesethylene glycol. In alternative embodiments, the denaturant is acombination of glycerol (e.g., 10%) and ethylene glycol (e.g., 6% to7%). Salts that reduce hybridization stringency may be included in thereaction buffers at low concentrations to chemically denature the targetDNA is at low temperatures. In embodiments where the target DNA isthermally denatured the denaturing step comprises thermally denaturingthe target DNA (e.g., by heating the target DNA at 95° C.).

In some embodiments, a DNA amplification method is provided, wherein atarget DNA is hybridized with an inosine-containing primer followed byamplification of the target DNA using a DNA polymerase (e.g., a stranddisplacement polymerase or a reverse transcriptase),deoxyribonucleotides (dNTPs) and an endonuclease. The dNTPs provides acombination of deoxyribonucleotides required by a DNA polymerase for DNAsynthesis. DNA polymerases use dNTP mixture to add nucleotides to the 3′end of a primer based on a template strand of DNA in a complementaryfashion, creating a new DNA strand complementary to the target DNAtemplate. The dNTP mixture may include each of the naturally occurringdeoxynucleotide bases (i.e., adenine (A), guanine (G), cytosine (C), andThymine (T)). In some embodiments, each of the naturally occurringdeoxynucleotides may be replaced or supplemented with a syntheticanalog; provided however that deoxyinosinetriphosphate may not replaceor supplement dGTP in the dNTP mixture. The product of DNA amplificationreaction may be single stranded or double-stranded DNA, often extendingto the end of the template strand. The inosine nucleotide in theinosine-containing primer may be positioned at least 4 nucleotides, atleast 5 nucleotides, or at least 10 nucleotides from the 5′ end of theinosine-containing primer. In certain embodiments, the inosinenucleotide may be the penultimate 3′ nucleotide of the primer. Inalternative embodiments, inosine may be present at both the penultimate3′ residue and ultimate 3′ residue. In some embodiments, theinosine-containing primer comprises an inosine analogue.

In some embodiments, an endonuclease, in combination with a stranddisplacing DNA polymerase and an inosine-containing primer is used foramplification of a target DNA (see FIG. 1 for a schematic representationof a nucleic acid amplification). Upon binding of inosine-containingprimer to the target DNA, the DNA polymerase (e.g., Phi29 DNApolymerase) extends the inosine-containing primer, generating a doublestranded DNA (primer extension product) and thereby creating a nickingsite for the endonuclease. The endonuclease nicks the double strandedDNA at this nicking site. Nicking creates a DNA synthesis initiationsite for the DNA polymerase. The DNA polymerase binds to this initiationsite and further elongates the nicked primer, displacing asingle-stranded DNA product while it recreates the double-strandedprimer extension product. The cycle repeats, synthesizing multiplesingle strands of DNA complementary to the downstream portion of thetarget DNA template.

The schematic representation of a nucleic acid amplification shown inFIG. 1 may be varied by employing additional primers or otheroligonucleotides, additional enzymes, additional nucleotides, stains,dyes, or other labeled components. For example, amplification with asingle primer may be used for dideoxy sequencing, producing multiplesequencing products for each molecule of template, and, optionally bythe addition of dye-labeled dideoxynucleotide terminators. Labeledprobes may be generated from double-stranded cDNA made with asequence-tagged oligo dT primer from mRNA samples. A single primer maybe the complement of the tag sequence, facilitating identificationand/or isolation.

In some embodiments, a strand displacement DNA polymerase, anendonuclease V and inosine-containing primers are employed in a DNAamplification reaction. Endonuclease V is a repair enzyme thatrecognizes DNA containing inosines (or inosine analogues) and hydrolyzesthe second or third phosphodiester bonds 3′ to the inosine (i.e.,specifically nicks a DNA at a position two nucleotides 3′ to an inosinenucleotide) leaving a nick with 3′-hydroxyl and 5′-phosphate. When thetarget DNA is double stranded the nick occurs in the strand comprisingthe inosine residue. For DNA amplification, the inosine-containingprimer hybridizes with the target DNA. Inosine residue in the primer maybase pair with a cytidine residue or a thymidine residue in the targetDNA, wherein hypoxanthene substitutes for a guanine to complement acytosine; or substitutes for an adenine to complement a thymine Acomplimentary strand to the target DNA template is then generated by DNAsynthesis thereby generating a double-stranded DNA. Generation of thedouble stranded DNA in turn generates a nicking site for theendonuclease V. The endonuclease V nicks the inosine-containing strandof this double-stranded DNA. The DNA polymerase repeatedly generates thecomplementary strand from the nicked position. In each cycle, the stranddisplacement DNA polymerase employed in these reactions displace thecomplementary strand that was generated in the previous cycle. The stepsof hybridization, elongation, nicking and further elongation may occursubstantially simultaneously. Thus, one or more embodiments of thepresent invention provides methods wherein an inosine residue isintroduced into a specific position of a target nucleic acid (via anoligonucleotide primer), followed by repeated generation ofcomplimentary strand of the target nucleic acid using a polymerase andan endonuclease V that nicks the generated double stranded nucleic acidat the inosine-containing strand to initiate a second cycle ofcomplementary strand generation by the polymerase.

In some embodiments, the DNA amplification reaction is performed underisothermal conditions. The reaction temperature during an isothermalamplification reaction condition may range 1° C., 5° C., or 10° C. froma set temperature. In some embodiments, the reaction temperature of DNAamplification is held at 46° C. (±1° C.). Thermally stable endonucleasesand thermally DNA polymerases may be used depending upon the reactiontemperature of DNA amplification reaction.

Inosine-containing primers may be synthesized using any of theart-recognized synthesis techniques. Amplicons may be generated using asingle inosine-containing primer, paired inosine-containing primers, ornested-paired inosine-containing primers. Primer design software such as“autodimer” may be employed to design a single primer or multipleprimers capable of annealing to a nucleic acid and facilitatingpolymerase extension. The inosine-containing primer may be designed insuch a way that the melting temperature of the primer is about 45° C.with a salt concentration of about 50 mM. In some embodiments,relatively short primers (e.g., 10-mers to 20-mers; more preferably14-mers to 18-mers, most preferably 16-mers) may be employed.

In some embodiments, the inosine-containing primer is designed such thatthe inosine residue is positioned in the primer at a locationcomplementary to a Cytosine base (C) in the target DNA. In someembodiments, the inosine appears as the penultimate 3′ base of theprimer. Because the reaction conditions (i.e., temperature and ionicstrength) affect annealing of primer to target DNA, optimal positioningof the inosine in the primer may be adjusted according to the reactionconditions. In general, the inosine residue is positioned away from the5′ end of the prime such that the primer remains annealed to the targetDNA after nicking by the endonuclease (i.e., the length of the nickedprimer is sufficient to enable binding to the target DNA under thenucleic acid assay reaction conditions). Accordingly, the segment of theprimer 5′ of the inosine should have a melting temperature approximatelyequal to the reaction temperature at the chosen reaction conditions. Insome embodiment, the inosine-containing primer may comprise more thanone inosine residue or inosine analogues. If there are two template Gsin a row, two inosines may appear in the primer as the both thepenultimate 3′ and the final residues. In this case, nicking by theendonuclease 2 nucleotides 3′ to either inosine residues would have thesame effect of creating a nicked DNA strand. In some embodiments, theinosine-containing primer may demonstrate a melting temperature of 25°C. to 70° C., 30° C. to 65° C., or 40° C. to 55° C. in the reactionmixture. In some embodiments, the inosine-containing primer demonstratesa melting temperature of 45° C. in the reaction mixture.

With a single, forward primer, the rate of synthesis of complimentarycopies of target DNA is relatively constant, resulting in a steady,linear increase in the number of complimentary copies with time.Multiple primers may be included in the reaction mixture in someembodiments to accelerate the amplification process. Embodiments whereboth the plus and minus strands are generated, paired primers comprisinga forward primer and a reverse primer may be included in the reactionmixture. For example, when a reverse primer (a primer that anneals tothe generated complementary strand ((+) strand) to further generate a(−) strand in the reverse direction) that anneals to the complementarystrand of target DNA at a defined distance from the forward primer isadded (see, for example, FIG. 2), amplification process is accelerated.Since the targets for each of these primers would be present in theoriginal template, both strands would be amplified in the two primerscheme (hereinafter referred as “Ping-Pong” reaction, “Ping product”being the amplicon of the forward primer and the “Pong product” beingthe amplicon of the reverse primer). The inclusion of multiple pairedprimers may improve the relative percentage of a discrete product in thereaction mixture. The forward and reverse primers may be placedrelatively close to each other (i.e., less than about 1 kb apart),minimizing the time required to complete the forward amplicon ((+)strand) to its 5′ end as defined by the endonuclease V cleavage site,and thereby reducing the total time required to generate amplicons fromthe target DNA. The reaction rate reaches a maximum when the amount ofnuclease, polymerase, or any other component(s) becomes limiting.Additional pairs of nested primers (see, for example, FIG. 3) may alsobe used to further increase amplification rates. Nested primers may bedesigned to bind at or near the 3′ end of the previous amplicon so thatin a series, each primer in the series will hybridize next to each otheron the original target. Where multiple nested primers are used, singlestranded DNA binding protein (SSB) at a concentration of 1 ng to 1 μg ina 10 μL volume may be included in the reaction mixture to increasefidelity and to reduce background.

Amplification with multiple, paired primers facilitates rapid andextensive amplification, which is useful to detect the presence ofspecific sequences, to quantify the amounts of those sequences presentin a sample, or to produce quantities of a sequence for analysis bymethods such as electrophoresis for size measurement, restriction enzymedigestion, sequencing, hybridization, or other molecular biologicaltechniques.

In some embodiments, extender templates, which are specific primersequences (e.g., Ones that generate a promoter sequence or a restrictionendonuclease site specific sequence), may be annealed at the 3′ end ofthe amplicon by incorporating in an inosine-containing primer. Anextender template may be designed such that it anneals to the 3′ end ofan amplicon. If the extender template contains two stretches ofsequences, one complementary to the amplicon, and one that is not,hybridization will create a 5′ overhang of the non-complementary primersequence. The 3′ recessed end of the amplicon can then be furtherextended by the DNA polymerase. This extension reaction may be employedto incorporate specific DNA sequences at the 3′ end of an amplicon.

In some embodiments, the 5′ end of the extender template may contain ahairpin loop, with a fluorescent dye and a quencher located on eitherarm of the stem such that the dye fluorescence is largely quenched byresonance energy transfer. Upon extension of the recessed 3′ end of theamplicon by a DNA polymerase, the stem-loop structure gets converted toa double stranded structure and causes the dye and the quencher to beseparated further. This eliminates some or all of the fluorescencequenching, and thus generates a detectable signal. This signal may bemultiplexed by appropriate sequence selection of the extender templatesand the color of the quenched dyes so that 2 or more independentamplification processes may be monitored simultaneously.

In some embodiments the 5′ end of the extender template may include thecomplement of an RNA polymerase promoter sequence. Thus, a doublestranded RNA polymerase promoter may be generated by hybridization ofextender template to the amplicon followed by extension of the recessed3′ end of the amplicon by the DNA polymerase. If an RNA polymerase isincluded in the reaction, the amplicon may be then transcribed as asingle-stranded RNA polymerase template to generate corresponding RNAs.

The nucleic acids produced by various embodiments of the present methodsmay be determined qualitatively or quantitatively by any of the existingtechniques. For example, for a qualitative or quantitative assay,terminal-phosphate-labeled ribonucleotides may be used in combinationwith a phosphatase during/after nucleic acid amplification reaction forcolor generation. In such embodiments, the terminal phosphate may beprotected from dephosphorylation by using terminal-phosphate methylesters of dNTPs or deoxynucleoside tetraphosphates.

Any of the DNA polymerases known in the art may be employed for DNAamplification. DNA polymerases suitable for use in the inventive methodsmay demonstrate one or more of the following characteristics: stranddisplacement activity; the ability to initiate strand displacement froma nick; and/or low degradation activity for single stranded DNA. In someembodiments, the DNA polymerase employed may be devoid of one or moreexonuclease activity. For example, the DNA polymerase may be a 3′→5′exonuclease-deficient DNA polymerase or the DNA polymerase may lack5′→3′ exonuclease activity. In some embodiments, the DNA polymerase maylack both 3′→5′ and 5′→3′ activity (i.e., an exo (−) DNA polymerase).Exemplary DNA polymerases useful for the methods include, withoutlimitation, Phi29 DNA polymerase, Klenow, 5′→3′ exonuclease-deficientBst DNA polymerase (the Klenow fragment of Bst DNA polymerase I), 5′→3′exonuclease-deficient delta Tts DNA polymerase (the Klenow fragment ofTts DNA polymerase I), exo (−) Klenow, or exo(−) T7 DNA polymerase (T7Sequenase).

Any of the art-recognized buffers for nucleic acid synthesis reactions(e.g., Tris buffer, HEPES buffer) that results in a reaction pH between6 and 9 may be used. In some embodiments, the pH of the nucleic acidamplification reaction is 7.7. In general, buffers that enhance DNAstability (e.g., HEPES) may be preferred in certain amplicon productionmethods. However, thermo labile buffers such as Tris:Borate, HEPES, andMOPS buffers may be disfavored for some specific amplicon productionmethods employing thermal denaturation of a target DNA.

Polymerase enzymes typically require divalent cations (e.g., Mg⁺², Mn⁺²,or combinations thereof) for nucleic acid synthesis. Accordingly, one ormore divalent cations may be added to the reaction mixture. For example,MgCl₂ may be added to the reaction mixture at a concentration range of 2mM to 6 mM. Higher concentrations of MgCl₂ may be preferred when highconcentrations (e.g., greater than 10 pmoles, greater than 20 pmoles, orgreater than 30 pmoles) of inosine-containing primer are included in thereaction mixture.

The reaction mixture for nucleic acid assay may further include one ormore surfactants (e.g., detergents). Surfactants may be applied to thereaction tube before introducing the first component of the reactionmixture. Alternatively, surfactants may be added to the reaction mixturealong with the reaction components. In some embodiments, the surfactantmay be a detergent selected from Tween-20, NP-40, Triton-X-100, orcombinations thereof. In some embodiments, 0.05% NP-40 and 0.005% TritonX-100 are added to the reaction mixture. In some specific embodiments,the reaction buffer may comprise 25 mM Tris:borate; 5 mM MgCl₂; 0.01%Tween; and 20% ethylene glycol.

One or more blocking agents such as an albumin (e.g., BSA or HSA) may beadded to the reaction mixture to bind to the surface of the reactionvessel (e.g., plastic microcentrifuge tube or microtiter plate) therebyincreasing the relative amount target DNA that is available for reactionwith the nucleases or polymerases.

In some other embodiments reaction mixture may include at least onetopoisomerase (e.g., a type 1 topoisomerase). In some embodiments, thetopoisomerase may be present in the reaction mixture at a finalconcentration of at least 0.1 ng/μL.

In some embodiments, the reaction mixture may include at least onesingle stranded DNA binding protein (e.g., E. coli SSB, T4 gene 32protein (T4 g32p), T7 gene 2.5 protein, Ncp7, recA, or combinationsthereof). In some embodiments, at least one single stranded DNA bindingprotein may be present in the reaction mixture at a final concentrationof at least 0.1 ng/μL.

The reaction mixture may include one or more reducing agents (e.g.,dithiothreitol (DTT), 2-mercaptoethanol (βME), Tris(carboxyethyl)phosphine (TCEP), or 2-mercaptoethylamine (MEA)) that reduces theoxidation of enzymes in the reaction mix and improves the quality andyield of the amplicons produced.

In some embodiments, a wild type endonuclease V may be employed fornucleic acid assays that involves a nicking of an inosine-containing DNAas described in the inventive methods. Non-limiting examples of wildtype enodonulease V includes endonuclease V from Escherichia,Archaeoglobus, Termotoga, Salmonella, Yersinia or human. For example,the wild type endonuclease V may be an E. coli endonuclease V (SEQ IDNO: 1), Afu endonuclease V (SEQ ID NO: 56) or a Tma endonuclease V (SEQID NO: 57). Afu endonuclease V may be preferred in assays whereinhighest level of specificity for inosine-containing strand nicking sinceAfu endonuclease V is very specific for inosine and does not act onother abasic or hairpin type of structures.

In some embodiments, a mutant endonuclease V is employed to nick theinosine-containing DNA. The mutant endonuclease V may be generated byany of the art-recognized techniques for genetic engineering/proteinengineering of proteins including site-directed mutagenesis orartificial gene synthesis. The genetic engineering may include analteration of one or more amino acid residues of a wild typeendonuclease V. The alteration may include substitution, insertionand/or deletion of one or more amino acid residues of the wild typeendonuclease V. Mutant endonuclease V may be generated by rationaldesign of protein or by directed evolution. In some embodiments, arationally designed, mutant endonuclease V enzyme is employed that hasincreased substrate binding, increased nicking efficiency, increasednicking specificity and/or increased nicking sensitivity. A mutantendonuclease V may also be designed such that the substrate binding isreversible. The mutant endonuclease V enzyme may then support repeatednicking by each enzyme, whereas the corresponding wild type enzyme iscapable of only a single round (or a few limited rounds) of nicking (forexample, the wild type E. coli endonuclease V remains bound to the DNAafter nicking). Such mutant endonuclease V may be used in a reactionmixture in less than stoichiometric quantities to effect a nickingreaction. For example, FIG. 11B demonstrates a repeated nicking of amutant E. coli endonuclease V enzyme leading to exponential nickingreaction kinetics with time.

In some embodiments, a mutant E. coli endonuclease V is provided. Insome embodiments, the mutant E. coli endonuclease is a Y75A mutant E.coli endonuclease V corresponding to SEQ ID NO: 2. This mutant isgenerated by replacing the Tyrosine (Y) residue at the 75^(th) positionof a wild type E. coli endonuclease V (SEQ ID NO: 1) with an Alanine (A)residue. In some embodiments, a mutant Afu endonuclease Y74A (SEQ IDNO:3) and/or its conservative variants is employed. The mutant Y74A Afuendonuclease is generated by substituting a Tyrosine (Y) residue at the75^(th) position of a wild type Afu endonuclease V (SEQ ID NO: 56) withan alanine (A) residue.

In some embodiments, conservative variants of the mutant endonuclease Vare provided. For example, further alteration of a mutant endonuclease Vvia substitution, deletion, and/or addition of a single amino acid or asmall number (typically less than about ten) of amino acids may be a“conservative variant” if the physico-chemical properties of the alteredmutant endonuclease V is similar to the original mutant endonuclease V.In some cases, the alteration may be a substitution of one amino acidwith a chemically similar amino acid.

In some embodiments, a heat stable endonuclease V is preferred. Forexample, in a nucleic acid assay, where thermal denaturation (eitherpartial or full denaturation) of a target DNA is performed, a heatstable endonuclease V or a heat stable endonuclease V mutant may bepreferred. In other embodiments where thermal denaturation of a targetDNA is not required, a wild type endonuclease V or an endonuclease Vmutant (e.g., Y75A mutant E. coli endonuclease V) that has maximumenzymatic activity at a relatively low temperature (e.g., 45° C.) may bepreferred. For example, Y75A E. Coli endonuclease V mutant isinactivated by incubation at 50° C., whereas it retains its enzymaticactivity at 37-40° C. Afu endonuclease V (both wild type and Y75Amutant) or Tma endonuclease V (both wild type and Y80A mutant) aregenerally more thermo stable than the E. coli endonuclease V (both wildtype and Y75A mutant). In some embodiments where strand displacement DNAsynthesis by DNA polymerase may be increased by incubation at anelevated temperature, an endonuclease V which functions at hightemperature (e.g., 45-80° C.) may be preferred.

In some embodiments, mutant endonuclease V preferentially nicks theinosine-containing strand of a double stranded DNA at a position 3′ tothe inosine residue when the inosine residue is paired with a cytosineresidue. In some other embodiments, endonuclease V mutant preferentiallynicks the inosine-containing strand of a double stranded DNA at aposition 3′ to the inosine residue when the inosine residue is pairedwith a thymine residue. The mutant endonuclease V may have a higherefficiency than the wild type endonuclease V to nick theinosine-containing strand of the double stranded DNA when the inosine ispaired with cytosine or thymine. Further, a mutant endonuclease V maypreferentially nick an inosine-containing strand of a double strandedDNA than an inosine-containing single stranded DNA. For example, Y75A E.coli mutant endonuclease V nicks a double stranded DNA comprising aninosine residue better than a single stranded DNA comprising an inosineresidue. In contrast, Y80A Tma mutant endonuclease V nicks a singlestranded DNA comprising an inosine residue better than a double strandedDNA comprising an inosine residue. Some mutant endonucleases may nickstructures other than DNA sequences containing inosine residue whilesome others may be very specific to inosine-containing DNA sequences.For example, Tma and Afu endonucleases does not nick structures such asflaps and pseudo Y structures. In some embodiments, when there aremultiple inosine residues in a double stranded DNA, the endonuclease Vmutant may preferentially nick (often 1 or 2 nucleotides 3′ to theinosine residue) the inosine residue that is paired with a cytosineresidue than the inosine residue that is paired with a thymine residue.In some aspects, the endonuclease V mutant may nick a double strandedDNA containing base pair mismatches. The nicking may happen at thelocation of the base pair mismatch or at a location 3′ to the base pairmismatch that is separated by one or more bases.

In some embodiments, an isolated nucleic acid sequence comprising asequence that encodes the mutant endonuclease V is also provided. Insome embodiments, the isolated nucleic acid comprises a nucleic acidsequence of SEQ ID NO: 59 or of a degenerate variant of SEQ ID NO: 59.The isolated nucleic acid sequence may comprise a sequence that encodesa polypeptide consisting of the amino acid sequence of SEQ ID NO: 2(Y75A mutant E. coli endonuclease V). In some other embodiments, theisolated nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 60or of a degenerate variant of SEQ ID NO:60. The isolated nucleic acidsequence may comprise a sequence that encodes a polypeptide consistingof the amino acid sequence of SEQ ID NO: 3 (Y75A mutant Afu endonucleaseV). The isolated nucleic acid sequence may further be incorporated intoa suitable vector, may be delivered to a host cell (for example, viatransfection, transduction or transformation) and may be expressed togenerate the desired mutant endonuclease V. The expressed mutantendonuclease V may be isolated and purified from the host cell byemploying any of the art-recognized techniques for protein isolation andpurification.

In some embodiments, a nucleic assay that employs the mutantendonuclease V is provided. The nucleic acid assay may include any assaythat involves selective nicking of a double stranded DNA by a mutantendonuclease V at a position 3′ to an inosine residue, when the inosineresidue is base-paired with a cytosine residue. In some embodiments, theassay includes the steps of (a) providing a double stranded DNAcontaining an inosine residue paired with a cytosine residue (b) nickingthe double stranded DNA at a residue 3′ to the inosine residue and (c)performing the nucleic acid assay. The double stranded DNA containingthe inosine residue base-paired with a cytosine residue may be generatedby hybridizing an appropriately designed inosine-containing primer to atarget DNA template, wherein the target DNA template is a singlestranded DNA sequence. In some embodiments, the double stranded DNAcontaining inosine residue paired with cytosine residue is generated insitu by a primer-mediated replication of a target DNA template. Thenucleic acid assay may be a nucleic acid amplification reaction, anucleic acid detection reaction or both. In some embodiments, a nucleicacid amplification is performed by extending the nicked strand (thestrand containing the inosine residue) via a nucleic acid extensionreaction using a DNA polymerase and dNTPs. The assay may be performed byusing any mutant endonuclease V from any source that is capable ofnicking an inosine-containing strand of a double stranded DNA whereinthe inosine reside is base-paired with a cytosine residue. In someembodiments, the DNA polymerase employed may be devoid of one or moreexonuclease activity. For example, the DNA polymerase may be a 3′→5′exonuclease-deficient DNA polymerase or the DNA polymerase may lack5′→3′ exonuclease activity (proofreading activity). In some embodiments,the DNA polymerase may lack both 3′→5′ and 5′→3′ activity (i.e., an exo(−) DNA polymerase). In some embodiments, a strand displacement DNApolymerase is used for the nucleic acid extension reaction. ExemplaryDNA polymerases useful for the methods include, without limitation, BstDNA polymerase, delta Tts DNA polymerase, Klenow, 5′→3′exonuclease-deficient Bst DNA polymerase, 5′→3′ exonuclease-deficientdelta Tts DNA polymerase, exo (−) Klenow, exo (−) Bst DNA polymerase orexo (−) delta Tts DNA polymerase. In some embodiments, the nucleic acidextension reaction is performed under isothermal conditions.

The nucleic acid amplification methods described herein may be used toamplify genomes or fragments of genome for subsequent SNP analysis. Itcould also be used to generate antisense probe from mRNA using an oligo(dT) primer containing an inosine residue near the end. Combined withPhi29 DNA polymerase the DNA amplification methods may be employed toamplify extremely large pieces of DNA. The methods may also be used forlinear amplification of one strand or exponential amplification of bothstrands of a target DNA.

In some embodiments, the nucleic acid assay that employs the mutantendonuclease V comprises a single tube DNA amplification and sequencing.Single tube DNA amplification and sequencing may use a combination ofplasmid amplification and cycle sequencing. For example, a DNApolymerase such as Phi29 DNA polymerase, random hexamers and dNTP's maybe employed for amplification of a plasmid. A thermally stablesequencing DNA polymerase (which does not work well at 30° C.), 3′charged dye labeled terminators (which are not incorporated by phi29 DNApolymerase), and a sequencing primer which is thiophosphorylated at the3′ end to prevent degradation by the phi29 DNA polymerase may beincluded during the amplification reaction. After a short period at 30°C. to allow the plasmid to be amplified, the temperature of the reactionmixture may be raised and cycled from 95 to 60° C. The 3′ chargedterminators are important for allowing the use of dGTP in the sequencingreaction, as they prevent secondary structures during electrophoresis.In this reaction, the amplification components do not affect thesequencing reaction (the phi29 DNA polymearase is heat inactivated andthe hexamers do not bind at temperatures over 45° C.) and the sequencingcomponents do not affect the amplification reaction (the DNA polymeraseis inactive at low temperatures and the terminators are not incorporatedby the phi29 DNA polymerase). However, under such reaction conditions,the sequencing primer may get extended during the amplificationreaction. To avoid such sequencing primer extension reaction, the mutantendonuclease V may be used in such single tube DNA amplification andsequencing reactions. For example, the sequencing primer may be designedto comprise an inosine residue located near to its 3′ end (e.g., atabout 3 nucleotides from its 3′ end), and a thiophosphate bond on its 3′terminal base. Due to the thiophosphate bond on its 3′ terminal end, theprimer will not get degraded during the amplification reaction. Further,if the primer is getting extended during the amplification reaction, themutant endonuclease V would nick the extended primer back to originalsize thus making it available for subsequent sequencing reaction. Athermo labile mutant endonuclease V may be selected for the reactionsuch that the endonuclease V gets inactivated during the first cyclealong with the phi29 DNA polymerase. Once these enzymes are inactivated,subsequent sequencing reaction may be conducted in the same tube.

The mutant endonuclease V enzymes described herein may also be othernucleic acid assays. For example, they may be used to remove primersafter PCR or other types of amplification that contains such primers.They may be employed to clip a DNA sequence that is attached to asurface by a segment of DNA containing an inosine residue. Further, theymay be used to amplify one strand of a PCR product that contains aninosine residue in one of its primers. Other applications of such mutantendonuclease V enzymes include, but not limited to, to degrade DNA thatwas produced in a reaction containing d-inosine-triphosphate and todegrade PCR primers after a PCR reaction, in which the primers used forPCR contained inosine replacing the guanosine.

In some embodiments, a kit for nucleic assay comprising an endonucleaseV is provided. The endonuclease V may be a wild type endonuclease or amutant endonuclease V. In some other embodiments, a kit comprising agenetically engineered, mutant endonuclease V is provided. The mutantendonuclease V may be a protein the sequence of which consists of, SEQID NO: 2, SEQ ID NO: 3, or conservative variants thereof. In someembodiments, the mutant endonuclease may be a Y75A mutant E. coliendonuclease V, or a Y75A mutant Afu endonuclease V.

In some embodiments, an amplicon production kit is provided thatcomprises at least one inosine-containing primer, at least one DNApolymerase and at least one nuclease that is capable of nicking DNA at aresidue 3′ to an inosine residue. The DNA polymerase may be anexonuclease-deficient DNA polymerase. In some embodiments, the DNApolymerase may be an exonuclease-deficient DNA polymerase with stranddisplacement activity. In some embodiments, the nuclease that is capableof nicking DNA at a residue 3′ to an inosine residue may be anendonuclease V. In some embodiments, the kit comprises a mutantendonuclease V, the sequence of which consists of, SEQ ID NO: 2, SEQ IDNO: 3, or conservative variants thereof. The mutant endonuclease may bea Y75A mutant E. coli endonuclease V, or a Y75A mutant Afu endonucleaseV.

The amplicon production kit may further comprise a chemical denaturant(e.g., glycerol, ethylene glycol, or formamide) and may further includea surfactant (e.g., Tween-20, NP-40, Triton-X-100, or a combinationthereof). The amplicon production kit may further include one or moredivalent cations (e.g., Mn²⁺, Mg²⁺, or a combination thereof), which maybe present in the buffer at a final concentration of 2 mM to 6 mM. Theamplicon production kit may further comprise a reducing agent (e.g.,DTT, βME, MEA, or TCEP) and/or at least one single stranded DNA bindingprotein (e.g., E. coli SSB, T4 gene 32 protein, T7 gene 2.5 protein,Ncp7, recA, or combinations thereof). In some embodiments, the ampliconproduction kit may further comprise at least one at least one blockingagent comprising albumin and/or at least one topoisomerase.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the scope of the present inventionas defined by the appended claims. Some abbreviations used in theexamples section are expanded as follows: “mg”: milligrams; “ng”:nanograms; “pg”: picograms; “fg”: femtograms; “mL”: milliliters;“mg/mL”: milligrams per milliliter; “mM”: millimolar; “mmol”:millimoles; “pM”: picomolar; “pmol”: picomoles; “μL”: microliters;“min.”: minutes and “h.”: hours.

Commercially available stock buffers used in Examples are shown inTables 1-3 below. ThermoFidelase was obtained from Fidelity Systems,Gaithersburg, Md.; T7, DEPC water, and NaCl were obtained from Ambion;dNTPs were obtained from GE Healthcare; Tris-HCl and Tween 20 wereobtained from Sigma Aldrich. Volumes shown in the following Tables arein microliters unless otherwise indicated

TABLE 1 10X Sequenase Buffer Reagent Volume Concentration 1M Tris-HCl,pH 7.6 800 μL  400 mM 1M MgCl₂ 400 μL  200 mM 5M NaCl 200 μL  500 mM 100mM dATP  50 μL  2.5 mM 100 mM dCTP  50 μL  2.5 mM 100 mM dGTP  50 μL 2.5 mM 100 mM dTTP  50 μL  2.5 mM Tween 20  2 μL 0.1% 1M DTT  20 μL  10 mM DEPC Water 378 μL Total Vol. 2000 μL 

TABLE 2 10X Klenow Buffer Reagent Volume Concentration 1M Tris-HCl, pH7.6 1000 μL 0.5M 1M MgCl₂  200 μL 0.1M 100 mM dATP  50 μL 2.5 mM 100 mMdCTP  50 μL 2.5 mM 100 mM dGTP  50 μL 2.5 mM 100 mM dTTP  50 μL 2.5 mMTween 20   2 μL 0.1% 1M DTT  20 μL  10 mM DEPC Water  578 μL TotalVolume 2000 μL

TABLE 3 10X Endonuclease V Buffer   100 mM Tris-Borate pH 8 0.1% Tween20 30 mM MgCl₂ 2.5 mM each dNTP 10 mM DTT

Table 4 provides the sequences of wild type endonucleases, mutantendonuclease V enzymes, template DNAs, and various primers that are usedin the examples. Bacillus cereus strain ATCC 15816 DNA gyrase subunit Agene used as a template is identified as DNAG5 in the followingexamples.

TABLE 4 Sequences of wild type endonucleases,mutnat endonucleases, template DNAs, and various primersSequence (N-term- Ref. No. C-term; 5′→3′) Length WT E. coli SEQ IDMIMDLASLRAQQIELAS 223 endonuclease V NO: 1 SVIREDRLDKDPPDLIAGADVGFEQGGEVTRAAM VLLKYPSLELVEYKVAR IATTMPYIPGFLSFREY PALLAAWEMLSQKPDLVFVDGHGISHPRRLGVAS HFGLLVDVPTIGVAKKR LCGKFEPLSSEPGALAP LMDKGEQLAWVWRSKARCNPLFIATGHRVSVDSA LAWVQRCMKGYRLPEPT RWADAVASERPAFVRYT ANQP Y75A mutantSEQ ID MIMDLASLRAQQIELAS 225 E. coli NO: 2 SVIREDRLDKDPPDLIAendonuclease V GADVGFEQGGEVTRAAM VLLKYPSLELVEYKVAR IATTMPAIPGFLSFREYPALLAAWEMLSQKPDLV FVDGHGISHPRRLGVAS HFGLLVDVPTIGVAKKR LCGKFEPLSSEPGALAPLMDKGEQLAWVWRSKAR CNPLFIATGHRVSVDSA LAWVQRCMKGYRLPEPT RWADAVASERPAFVRYTANQPLE Y74A mutant SEQ ID MLQMNLEELRRIQEEMS 221 Afu NO: 3RSVVLEDLIPLEELEYV endonuclease V VGVDQAFISDEVVSCAV KLTFPELEVVDKAVRVEKVTFPAIPTFLMFREGE PAVNAVKGLVDDRAAIM VDGSGIAHPRRCGLATY IALKLRKPTVGITKKRLFGEMVEVEDGLWRLLDG SETIGYALKSCRRCKPI FISPGSYISPDSALELT RKCLKGYKLPEPIRIADKLTKEVKRELTPTSKLK Ban 1 Template SEQ ID CAATTGTAATTTCTGTA 77 NO: 4CGTCTCTTATCATTGAA GCGCTCTTTTACTTCTG TTAATTCTTCACGAATA ATCTCAAGA P-1SEQ ID TCTTGAGATTATTCIT 15 NO: 5 P2-1 SEQ ID CAATTGTAATTTCTIT 15 NO: 6P3 SEQ ID AAATTAATACGACTCAC 51 NO: 7 TATAGGGTGAAGAATTA ACAGAAGTAAAAGAGCddC P-3 Mismatched SEQ ID  AAATTAATACGACTCAC 51 NO: 8 TATAGGGTTGAAGAATTAACAGAAGTAAAAGAGA P-3 NO ddC SEQ ID AAATTAATACGACTCAC 41 NO: 9TATAGGGTGAAGAATTA ACAGAAG P-3SD Mod SEQ ID AAATTAATACGACTCAC 51 NO: 10TATAGGGTTGAAGAATT AACAGAAGTAAAAGAGd dC Primer 1354 SEQ IDTCGCTGAATTAAAAIC 16 NO: 11 Primer 1333 SEQ ID ATCAAGATTTAATGAAI 18NO: 12 T Primer 1498 SEQ ID TGTTCTGGAATCAAIT 16 NO: 13 Primer 1517SEQ ID AACGTAATGGCGATIT 16 NO: 14 Primer 1275 SEQ ID TTAGATATGCGTCTIC 16NO: 15 Primer 1294 SEQ ID GCTTAACAGGATTAIA 16 NO: 16 Primer 1313  SEQ IDCGAAAAAATTGAACAAI 18 NO: 17 A Primer 1378 SEQ ID CAGATGAAGAAAAGIT 16NO: 18 Primer 1482 SEQ ID CTTCATCTTCAATAIA 16 NO: 19 Primer 1534 SEQ IDAATATAACCATTATGAI 18 NO: 20 T Primer 1560 SEQ ID TACGTAGAAGCTGIC 15NO: 21 Primer 1579 SEQ ID ACCACGGTTCTGTIT 15 NO: 22 cP-3 3′ SEQ IDCCCTATAGTGAGTCGTA 24 Quencher NO.: 23 TTAATTT-IowaBlack FluorescentSEQ ID FAM-GGTCGACTIAGGA 27 Primer 1 NO: 24 GGATCCCCGGGTAC FluorescentSEQ ID HEX-CCGGGGATCCTCC 27 Primer 2 NO: 25 TCAGTCGACCTGCA endoV usSEQ ID GAGATATACATATGGAT 23 NO: 26 CTCGCG endoV int ds SEQ IDGAATCGCCGGCATGGTG 28 NO: 27 GTGGCGATGCG endoV int us SEQ IDACCATGCCGGCGATTCC 33 NO: 28 AGGTTTTCTTTCCTTC endoV ds SEQ IDTGGTGCTCGAGGGGCTG 25 NO: 29 ATTTGATG DNA-G-Long-3′ SEQ IDGATATTCATCAATCGGA 27 NO: 30 GTACGTTTTC DNA-G-5′ SEQ ID ACAATCAACAACAAGCA 27 NO: 31 CGAATTCGAG 7290R SEQ ID AGTTCTTCTTTCGTCCC 20NO: 32 CIT 7270R SEQ ID CAGGCTGACATCACIIT 17 NO: 33 7253R SEQ IDTCAGTTGTTCACCCAGC 19 NO.: 34 IA 7234R SEQ ID GCGGAGACGGGCAATCA 19 NO: 35IT 7215R SEQ ID TCATCTTTCGTCATIIA 17 NO: 36 7194R SEQ IDTCCACAGAGAAACAATI 19 NO.: 37 IC 7062F SEQ ID ACCACCGGCGATCCIIC 17 NO: 387079F SEQ ID GCGTGAGTTCACCATIA 17 NO: 39 7096F SEQ ID TTCAGTCAGCACCGCTI18 NO: 40 A 7111F SEQ ID TGATGCTGCTGGCTIA 16 NO: 41 7127F SEQ IDCCCTGATGAGTTCGTIT 17 NO: 42 7144F SEQ ID CCGTACAACTGGCIT 15 NO: 43T7-7158F- SEQ ID ATGACTGGTGGACAGCA 50 misA: NO: 44 AATGGGTAAATTAATACGACTCACTATAGGGTT Quencher-oligo  SEQ ID CCCTATAGTGAGTCGTA 24 NO: 45TTAATTT-3iaBrqsP Dye-oligo SEQ ID ACCCAT/i6-TAMN/TT NO: 46GCTGTCCACCAGTTAC F. primer SEQ ID GTTTTCCCAGTCACGAC 34 40FGI NO: 47GTTGTAAAACGACGICC R. primer SEQ ID TCAAAGAAGTATTGCTA 23 40RGI NO: 48CAACGG F. primer: SEQ ID GTTTTCCCAGTCACGAC 34 40FGG NO: 49GTTGTAAAACGACGGCC R. primer SEQ ID TCAAAGAAGTATTGCTA 23 40RGG NO: 50CAACGG Tban-1 SEQ ID GCAGATGAAGAAAAGGT 33 forward primer: NO: 51TCTTGAGATTATTCIT Dye-P-3 SEQ ID 5′-TAMRA Dye-AAAT 51 NO: 52TAATACGACTCACTATA GGGTTGAAGAATTAACA GAAGTAAAAGAGdd C-3′ Wild Type AfuSEQ ID MLQMNLEELRRIQEEMS 221 endonuclease V NO: 56 RSVVLEDLIPLEELEYVVGVDQAFISDEVVSCAV KLTFPELEVVDKAVRVE KVTFPYIPTFLMFREGE PAVNAVKGLVDDRAAIMVDGSGIAHPRRCGLATY IALKLRKPTVGITKKRL FGEMVEVEDGLWRLLDG SETIGYALKSCRRCKPIFISPGSYISPDSALELT RKCLKGYKLPEPIRIAD KLTKEVKRELTPTSKLK Wild Type Tma SEQ ID Y80Y 225 endonuclease V NO: 57 Y80A mutant SEQ IDMDYRQLHRWDLPPEEAI 225 Tma NO: 58 KVQNELRKKIKLTPYEG endonuclease VEPEYVAGVDLSFPGKEE GLAVIVVLEYPSFKILE VVSERGEITFPAIPGLL AFREGPLFLKAWEKLRTKPDVVVFDGQGLAHPRK LGIASHMGLFIEIPTIG VAKSRLYGTFKMPEDKR CSWSYLYDGEEIIGCVIRTKEGSAPIFVSPGHLM DVESSKRLIKAFTLPGR RIPEPTRLAHIYTQRLK KGLF Y75A mutantSEQ ID GTGATTATGGATCTCGC 678 E. coli NO: 59 GTCATTACGCGCTCAACendonuclease V AAATCGAACTGGCTTCT TCTGTGATCCGCGAGGA TCGACTCGATAAAGATCCACCGGATCTGATCGCC GGAGCCGATGTCGGGTT TGAGCAGGGCGGAGAAG TGACGCGAGCGGCGATGGTGCTGCTGAAATATCC CTCGCTTGAGCTGGTCG AGTATAAAGTTGCCCGC ATCGCCACCACCATGCCTTACATTCCAGGTTTTC TTTCCTTCCGCGAATAT CCTGCGCTGCTGGCAGC GTGGGAGATGNNNTCGCAAAAGCCGGATTTAGTG TTTGTCGATGGTCATGG GATCTCGCATCCTCGCC GTCTTGGCGTCGCCAGCCATTTTGGCTTATTGGT GGATGTGCCGACCATTG GCGTGGCGAAAAAACGG CTCTGCGGTAAATTCGAACCGCTCTCCAGCGAAC CGGGCGCGCTGGCCCCA CTGATGGATAAAGGCGA GCAGCTGGCCTGGGTCTGGCGCAGCAAAGCGCGC TGTAACCCGTTGTTTAT CGCTACCGGCCATCGGG TCAGCGTGGACAGCGCGCTGGCGTGGGTACAACG CTGCATGAAAGGCTATC GTCTGCCGGAGCCAACG CGCTGGGCGGACGCGGTGGCCTCGGAACGTCCGG CGTTCGTGCGCTATACA GCAAATCAGCCCTAA Y74A mutant SEQ IDGTGCTTCAAATGAATCT 666 Afu NO: 60 CGAAGAGCTGAGGAGGA endonuclease VTACAGGAGGAGATGTCC AGAAGTGTGGTTCTCGA AGACTTAATCCCTCTTG AAGAGCTTGAGTACGTTGTGGGTGTTGATCAGGC CTTTATCAGCGATGAGG TTGTCTCATGTGCGGTC AAGCTGACCTTTCCGGAACTGGAGGTTGTTGATA AAGCTGTGAGGGTTGAG AAGGTCACTTTCCCCNN NATCCCCACCTTTCTCATGTTCAGGGAGGGAGAG CCTGCAGTTAATGCGGT CAAAGGGCTTGTGGATG ACAGAGCGGCAATCATGGTTGATGGGAGCGGAAT TGCCCATCCGAGAAGGT GCGGGCTTGCAACATAC ATCGCCCTAAAGCTGAGAAAGCCGACTGTGGGGA TAACAAAGAAAAGGCTT TTTGGTGAGATGGTAGA GGTGGAAGATGGGCTTTGGAGGCTTTTAGATGGA AGTGAAACCATAGGCTA CGCCCTTAAAAGCTGCA GGAGGTGCAAACCAATCTTCATCTCACCGGGGAG TTACATATCTCCTGACT CAGCCTTGGAGCTGACG AGAAAGTGCCTTAAAGGCTACAAGCTTCCTGAGC CGATAAGAATCGCCGAC AAACTTACCAAGGAGGT TAAGAGGGAGTTGACTCCAACCTCAAAGCTTAAA TAA

Amplicons may be visualized and/or quantified using any ofart-recognized techniques (e.g., electrophoresis to separate species ina sample and observe using an intercalating dye such as ethidiumbromide, acridine orange, or proflavine). Amplicon production may alsobe tracked using optical methods (e.g., ABI Series 7500 Real-Time PCRmachine) and an intercalating dye (e.g., SYBR Green I). The ampliconsproduced in the following examples were visualized using electrophoresisor optical techniques.

Example 1: Preparation of a PCR Product from the B. cereus Genomic DNAthat is Used as Template for Amplification Reactions (Hereinafter, thisPCR Product is Referred as “DNAG5”)

Bacillus cereus (B. Cereus) strain 15816 from American Type CultureCollection (ATCC, Manassas, Va.) was grown according to the supplier'srecommendations. A lyophilized culture of B. cereus was re-suspended in400 μL of Nutrient Broth then transferred to 5.6 mL of Nutrient Brothand incubated overnight at 30° C. with shaking. The Genomic DNA (gDNA)was isolated from the overnight liquid culture using MasterPure™ GramPositive DNA Purification Kit (Epicentre® Biotechnologies, #GP1-60303)according to the manufacturer's instructions.

4×1.0 mL of overnight culture was pelleted by centrifugation in separate1.5 mL tubes. Each pellet was resuspended in 100 μL TE Buffer byvortexing vigorously and added to separate 0.65 ml tubes. The 1.5 mLtubes were rinsed with 50 μL each TE Buffer and the wash were combinedwith the appropriate 100 μL of resuspended pellet. 1 μL of Ready-LyseLysozyme was added to each resuspended pellet, mixed, and incubated at37° C. for 60 minutes. 150 μL Gram Positive Lysis Solution & 1 μLProteinase K was added to each tube and mixed. Samples were incubated at70° C. for 15 minutes, vortexing every 5 minutes. The samples were thencooled to 37° C. for 5 minutes and then placed on ice for 5 minutes. 175μL MPC Protein Precipitation Reagent was added to each tube. The debriswas pelleted at high speed in a microfuge for 10 minutes. Supernatantwas transferred to a clean 1.5 mL tube and the pellet was discarded. 1μL RNase A (5 μg/μL) was added to each tube and incubated 30 minutes at37° C. 500 μL 2-propanol was added to each tube and inverted 35 times tomix. The tubes were centrifuged 10 minutes at high speed in a microfugeto pellet the DNA. Each pellet was rinsed with 70% ethanol, dried, andresuspended in 35 μL TE buffer. 1 μL from each preparation was run on a1% agarose gel.

Approximately 100 ng of B. cereus gDNA was amplified in four separatereactions using PuReTaq Ready-To-Go PCR Beads (GE Healthcare) and 5 μMof each primer, DNA-G-Long-3′ (SEQ ID NO:30) and DNA-G-5′ (SEQ IDNO:31). Thermal cycling conditions were: (1) 95° C. for 5 minutes; (2)95° C. for 30 seconds; (3) 50° C. for 30 seconds; (4) 72° C. for 1minute. Steps 2-4 were repeated 31 times and the products were held at+4° C.

One-tenth of each completed reaction was analyzed by electrophoresisusing a 1% agarose gel. A 2181 base pair product was generated and usedas the target DNA. Following analysis, the four amplification reactionswere pooled and diluted in TE Buffer ±0.01% Tween 20 prior to use.

Example 2: DNA Amplification Employing Various Polymerases, SinglePrimer and Two Primers

Exo (−) T7 DNA polymerase (Sequenase), exo(−) Bst polymerase (Bst largefragment) and exo (−) Klenow fragment were compared according to thereaction scheme presented below in Table 5 (volumes are indicated inmicroliters). P-1 corresponds to SEQ ID NO: 5 and P2-1 corresponds toSEQ ID NO: 6. Reactions 1, 2, 7, and 8 containing the exo (−) Bstpolymerase were incubated at 43° C. for 3 hours. The remaining reactionscontaining exo (−) Klenow or T7 Sequenase were incubated at 37° C. for 3hours. Following storage at −20° C., the reactions were run on a 15%acrylamide (Invitrogen), 7M-urea gel. The results are shown in FIG. 4,wherein the 61′-mer and 45′-mer depicts the expected products. The “Pingproduct” was generated in almost all reactions. A small amount of both“Ping product” and the “Pong product” was generated by Bst polymerase,whereas a large amount of both “Ping product” and the “Pong product” wasgenerated by exo (−) Klenow. Columns 13 and 14 in Table 5 represent thepolymerase extension of only primers.

TABLE 5 ID Reagent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 10X 1 1 1 1 1 1 1 1Endonuclease V Buffer 10X TP 1 1 0.5 0.5 Buffer 10X T7 1 1 0.5 0.5Buffer 10X Klenow 1 1 0.5 Buffer Ban 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.50.5 0.5 0.5 0.5 0.5 0.5 Template P-1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.50.5 0.5 0.5 2 2 P2-1 0.5 0.5 0.5 0.5 0.5 0.5 T4 g32p 0.5 0.5 0.5 0.5 0.50.5 0.5 0.5 0.5 0.5 0.5 0.5 Ethylene 2 2 2 2 2 2 2 2 2 2 2 2 Glycol Bst0.5 0.5 0.5 0.5 0.5 0.5 T7 0.5 0.5 0.5 0.5 5 5 Klenow 0.5 0.5 0.5 0.5 1010 Endonuclease 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 V Water4.5 4.5 4.5 4.5 4.5 4.5 4 4 4 4 4 4 Total 10 10 10 10 10 10 10 10 10 1010 10 Volume

Example 3: Multiple Sets of Primers and Single Strand Binding Protein

4, 6, and 12 mixes of primers were combined as shown below in Tables6-13. The primer mixes were prepared so that one addition would add 10pmol [final concentration] of each oligos to the reaction mixture.

12 Mix:

TABLE 6 Oligo P-1 P2-1 1275 1294 1313 1333 1378 1482 1517 1534 1560 1579

6 Mix:

TABLE 7 Oligo P-1 P2-1 1333 1378 1482 1517

4 Mix:

TABLE 8 Oligo P-1 P2-1 1378 1482

Internal 4 Mix: The term “internal” represents that these primers wereinternal to the other primers used in other reactions:

TABLE 9 Oligo 1354 (645 pmol/uL) 1333 (681 pmol/uL) 1498 (732 pmol/uL)1517 (671 pmol/uL)

Reaction Scheme: DNAG5 was diluted 1:100 in TE buffer. 10× HEMT bufferincludes 100 mM HEPES (pH 8), 1 mM EDTA, 0.1% Tween 20, and 30 mM MgCl₂.

TABLE 10 ID Component 1 2 3 4 5 6 7 8 9 10 11 12 DNAG5 1:100 − − − − − −− + + + + + 12 mix + − − − − − − + − − − − 6 mix − + − − − − − − + − − −4 mix − − + − − + + − − + − − Internal 4 Mix − − − + + + + − − − + +SSB, 1 μg/uL + − − + − + − + − − + − SSB, 10 ng/uL − + + − + − + − + +− + 1354 (10 pmol/uL) − − − − − − − − − − − − 1333 (10 pmol/uL) − − − −− − − − − − − − 1498 (10 pmol/uL) − − − − − − − − − − − − 1517 (10pmol/uL) − − − − − − − − − − − − ID Component 13 14 15 16 17 18 19 20 2122 23 24 DNAG5 1:100 + + + + + + − − − − − − B. cereus gDNA (100 ng/uL)− − − − − − + + + + + + 12 mix − − − − − − + − − − − − 6 mix + + − − − −− + − − − − 4 mix − − + + + + − − + − + + Internal 4 Mix − − − − + + − −− + + + SSB, 1 μg/uL + + − − + + − − − − − + SSB, 10 ng/uL − + + + −− + + + + + − 1354 (10 pmol/uL) + − + − − − − − − − − − 1333 (10pmol/uL) + − − + − − − − − − − − 1498 (10 pmol/uL) − + + − − − − − − − −− 1517 (10 pmol/uL) − + − − − − − − − −

Bulk Denaturation Mixes: Volumes shown in Tables are in microlitersunless otherwise indicated.

TABLE 11 ID Component A (X5) B (X5) C (X15) D (X13) 10X HEMT Buffer 5 515 13 12 mix 2.5 — — —  6 mix — 2.5 — —  4 mix — — 7.5 — Internal 4 Mix— — — 6.5 Water 10 10 7.5 7.5 Total Volume 17.5 17.5 30 26

3.5 μL of ‘A’ were added to each of 1, 8, and 19; 3.5 μL of ‘B’ wereadded to each of 2, 9, and 20; 2.0 μL of ‘C’ were added to each of 3, 6,7, 10, 13, 14, 15, 16, 17, 18, 21, 23, and 24; and 2.0 μL of ‘D’ wereadded to each of 4, 5, 6, 7, 11, 12, 17, 18, 22, 23, and 24.

Denaturations:

TABLE 12 ID Component 1 2 3 4 5 6 7 8 9 10 11 12 DNAG5 1:100 — — — — — —— 1 1 1 1 1 Bulk Denaturation Mix A 3.5 — — — — — — 3.5 — — — — BulkDenaturation Mix B — 3.5 — — — — — — 3.5 — — — Bulk Denaturation Mix C —— 2 — — 2 2 — — 2 — — Bulk Denaturation Mix D — — — 2 2 2 2 — — — 2 21354 (10 pmol/uL) — — — — — — — — — — — — 1333 (10 pmol/uL) — — — — — —— — — — — — 1498 (10 pmol/uL) — — — — — — — — — — — — 1517 (10 pmol/uL)— — — — — — — — — — — — Water 1.5 1.5 3 3 3 1 1 0.5 0.5 2 2 2 Total Vol.5 5 5 5 5 5 5 5 5 5 5 5 ID Component 13 14 15 16 17 18 19 20 21 22 23 24DNAG5 1:100 1 1 1 1 1 1 — — — — — — B. cereus gDNA (100 — — — — — — 1 11 1 1 1 ng/uL) Bulk Denaturation Mix A — — — — — — 3.5 — — — — — BulkDenaturation Mix B 2 2 — — — — — 3.5 — — — — Bulk Denaturation Mix C — —2 2 2 2 — — 2 — 2 2 Bulk Denaturation Mix D — 1 — — 2 2 — — — 2 2 2 1354(10 pmol/uL) 1 — 1 — — — — — — — — — 1333 (10 pmol/uL) 1 — — 1 — — — — —— — — 1498 (10 pmol/uL) — 1 1 — — — — — — — — — 1517 (10 pmol/uL) — — —1 — — — — — — — — Water 5 5 — — — — 0.5 0.5 2 2 — — Total Vol. 5 5 5 5 55 5 5 5 5 5 5

Bulk Enzyme Mixes: 10 Rxn Buffer used in Table 13 includes 100 mM HEPES,30 mM MgCl2, 0.1% Tween 20, 2.5 mM each dNTP, and 10 mM TCEP.

TABLE 13 ID Component 1X Y(X10) Z(X18) 10X Reaction Buffer) 1 10 18 100%Ethylene Glycol 1 10 18 E coli SSB, 1 μg/uL 1 10 — E coli SSB, 10 ng/uL— — 18 ΔTts (20 U/) 1 10 18 Endonuclease V (40 pmol/) 0.075 0.75 1.35Water 0.925 9.25 16.65 Total Volume 5 50 90

5 μL of component “X” were added to each of reaction mixes 1, 4, 6, 8,11, 17, 18, and 24; 5 μL of “Z” were added to each of 2, 3, 5, 7, 9, 10,12, 13, 15, 16, 19, 20, 21, 22, and 23. All reaction mixtures wereincubated at 45° C. for 75 minutes and a 1/10th aliquot was analyzed ona 10% TB Urea gel, in which each of the oligo mixtures generated productof the expected sizes. None of the reactions performed on genomic DNAyielded expected products. One of the 4-primer mixes did not work whenthe 1 microgram of SSB was added, but did give the expected productswhen 1 ng of SSB was added on the PCR product template (DNAG5).

Example 4: Isothermal Amplification Using Internal 6 Oligo Mix (AmpliconProduction Using Multiple Sets of Nested Primers)

Selected oligos were removed from the 6 oligo mix to determine whicholigo or oligos caused the doublet band between 70 and 80 bases. Also,variations of divalent cation and single strand binding proteinconcentrations were tested. None of the tested variations altered thedoublet band, and one remaining possibility is that it may be caused byincomplete denaturation during electrophoresis.

Oligo Mixes:

TABLE 14 Oligo A OM B OM P-1 (972 pmol/uL)  1.03 μL — P2-1 (335 pmol/uL) 2.99 μL  2.99 μL 1333 (681 pmol/uL)  1.47 μL  1.47 μL 1378 (645pmol/uL)  1.55 μL  1.55 μL 1482 (661 pmol/uL) —  1.51 μL 1517 (671pmol/uL)  1.49 μL  1.49 μL TE + 0.01% Tween 20 41.47 μL 40.99 μL TotalVolume   50 μL   50 μL

Reaction Scheme: Rxn Buffer A (10× reaction buffer) used in Table 15includes 100 mM HEPES, 30 mM MgCl₂, 0.1% Tween 20, 2.5 mM each dNTP, and10 mM TCEP. 10 Rxn Buffer B used in Table 15 includes 100 mM HEPES, 60mM MgCl₂, 0.1% Tween 20, 2.5 mM each dNTP, and 10 mM TCEP.

TABLE 15 ID Component 1 2 3 4 5 6 7 8 9 10 11 12 13 14 DNAG5 1:10 − − −− + + + + − − − − + + DNAG5 1:100 − − − − − − − − + + + + − − 12 Mix + −− − + − − − + + − − + + 6 Mix − + − − − + − − − − + + − − A OM − − + − −− + − − − − − − − B OM − − − + − − − + − − − − − − Rxn Buffer A − − − −− − − − − − + + − − (3 mM) Rxn Buffer B + + + + + + + + + + − − + + (6mM) 30 mM Mg − − − − − − − − − − − − + − 60 mM Mg − − − − − − − − − − −− − + 90 mM Mg − − − − − − − − − − − − − − SSB, 1 ng/Rxn − − − − − − −− + − + − − − SSB, 1 μg/Rxn + + + + + + + + − + − + + + ID Component 1516 17 18 19 20 21 22 23* 24* 25* 26* 27* 28* DNAG5 1:10 + + + + − − − −− − − − − − DNAG5 1:100 − − − − − − − − − − − − − − B. cereus + − −− + + + + − − − − − − gDNA 100 ng/Rxn 12 Mix − − − − + − − − + + + − − −6 Mix − + + + − + − − − − − + + + A OM − − − − − − + − − − − − − − B OM− − − − − − − + − − − − − − Rxn Buffer A + − − − − − − − − − − − − − (3mM) Rxn Buffer B − + + + + + + + + + + + + + (6 mM) 30 mM Mg − + − − − −− − + − − + − − 60 mM Mg + − + − − − − − − + − − + − 90 mM Mg − − − + −− − − − − + − − + SSB, 1 ng/rxn + − − − − − − − − − − − − − SSB, 1μg/uL + + + + + + + + + + + + + *Extra MgCl₂ added to the reactionmixture to understand if increased Mg²⁺ improves the reaction.

No Template Control Denaturations: 10× HE Buffer includes 100 mM HEPES(pH*) and 1 mM EDTA.

TABLE 16 ID Component 1 2 3 4 23 10x HE Buffer   1 μL   1μL   1μL   1μL  1 μL DNAG51:10 — — — — — DNAG51:100 — — — — — 12 Mix 0.5 μL — — — 0.5μL  6 Mix — 0.5 μL — — — A OM — — 0.5 μL — — B OM — — — 0.5μL — 30 mMMgCl₂ — — — —   1 μL 60 mM MgCl₂ — — — — — 90 mM MgCl₂ — — — — — Water2.5 μL 2.5 μL 2.5 μL 2.5 μL 1.5 μL Total Volume   4 μL   4 μL   4 μL   4μL   4 μL ID Component 24 25 26 27 28 10X HE Buffer   1 μL   1μL   1μL  1μL   1 μL DNAG5 1:10 — — — — — DNAG5 1:100 — — — — — 12 Mix 0.5 μL0.5 μL — — —  6 Mix — — 0.5 μL 0.5 μL 0.5 μL A OM — — — — B OM — — — —30 mM MgCl₂ — —   1 μL — — 60 mM MgCl₂   1 μL — —   1 μL —

Bulk Denaturation Mixes:

TABLE 17 ID Component A (X6) B (X6) C (X3) D (X3) 10X HE Buffer  6 μL  6μL   3 μL   3 μL DNAG5 1:10  6 μL  6 μL — — DNAG5 1:100 — —   3 μL   3μL 12 Mix  3 μL — 1.5 μL — 3 Mix —  3 μL — 1.5 μL OM A — — — — OM B — —— — Water  9 μL  9 μL 4.5 μL 4.5 μL Total Volume 24 μL 24 μL  12 μL  12μL 4 μL “A” in each of 5, 13, 14, and 15 4 μL “B” in each of 6, 16, 17,and 18 4 μL “C” in each of 9 and 10 4 μL “D” in each of 11 and 12

Denaturations:

TABLE 18 ID Component 7 8 19 20 21 22 10X HE Buffer 1 μL 1 μL 1 μL 1 μL1 μL 1 μL DNAG5 1:10 1 μL 1 μL — — — — DNAG5 1:100 — — — — — — B. cereusgDNA 100 ng/uL — — 1 μL 1 μL 1 μL 1 μL 12 Mix — — 0.5 μL   — — — 3 Mix —— — 0.5 μL   — — OM A 0.5 μL   — — — 0.5 μL   — OM B — 0.5 μL   — — —0.5 μL   30 mM MgCl₂ — — — — — — 60 mM MgCl₂ — — — — — — 90 mM MgCl₂ — —— — — — Water 1.5 μL   1.5 μL   1.5 μL   1.5 μL   1.5 μL   1.5 μL  Total Volume 4 μL 4 μL 4 μL 4 μL 4 μL 4 μL

Bulk Enzyme Mixes:

TABLE 19 ID Component V (X2) W (X2) X (X2) Y (X8) Z (X21) 10X Rxn BufferA   2 μL   2 μL — — — (3 mM) 10X Rxn Buffer B — —   2 μL   8 μL   21 μL(6 mM) 100% Ethylene Glycol   2 μL   2 μL   2 μL   8 μL   21 μL E. coliSSB (1 ng/uL)   2 μL —   2 μL — — E. coli SSB (1 μg/uL) —   2 μL —   8μL   21 μL ΔTts (20 U/uL)   2 μL   2 μL   2 μL   8 μL   21 μL Endo V (40pmol/uL) 0.15 μL 0.15 μL 0.15 μL 0.6 μL  1.58 μL Water 3.85 μL 3.85 μL3.85 μL 7.4 μL 85.58 μL Total Volume   12 μL   12 μL   12 μL  40 μL  126 μL 6 μL “V” in 11 6 μL “W” in 12 6 μL “X” in 9 5 μL “Y” in 13-18(Additional amount of MgCl₂ added in 1 μL volume, see Table 16) 6 μL “Z”in each of 1-8, 10, and 19-28

All reactions were incubated at 45° C. for 75 minutes and applied to agel. FIG. 5 demonstrates that Ping-Pong reaction generated the expectedproducts, however, no oligo eliminations removed the unwanted 74nucleotide product.

Example 5: In Vitro Transcription (IVT)

Bulk Standard Isothermal Amplification Reaction: Template Primer Mixincludes 0.08 pmol of SEQ ID NO: 4; 2 pmol of SEQ ID NO: 5; 10 pmol ofSEQ ID NO: 6; and 18 pmol of SEQ ID NO: 7.

TABLE 20 Component 1X 3X 10X Endonuclease V Buffer 1 3 Template PrimerMix 0.5 1.5 Klenow (10 U/μL) 0.5 1.5 Endonuclease V (8 pmol/μL) 0.5 1.5ThermoFidelase 1 3 TAP/T4 g32p (0.005 U TAP; 0.2 0.5 1.5 μg T4g32P)Water 6 18 Total Vol. 10 30

3 μL of the 3× bulk reaction were added to 6 μL GLB (gel loading buffer,Ambion) II (Before isothermal amplification). The remaining 3× bulkreaction was incubated at 45° C. for 75 minutes. The reaction wasstopped by adding 2.7 μL, 110 mM EDTA. A 1-μL aliquot from the reactionwas added to 2 GLB II (After isothermal amplification).Beta-Mercaptoethanol (β-ME) was obtained from Sigma (cat. #104K0161). 1MTris-HCl, pH 8 was obtained from Ambion (cat. #105R055626A). The rNTPmixtures are shown in Table 21 below and the general reaction mixturefor in vitro transcription is shown below in Table 22. For this example,components of MEGAscript T7 Kit (Ambion) were used.

TABLE 21 Component Amt 75 mM ATP 15 μL 75 mM CTP 15 μL 75 mM GTP 15 μL75 mM UTP 15 μL Total Volume 60 μL

TABLE 22 Component Amt Water  3 μL rNTP Mix  4 μL 10X Buffer  1 μLTemplate  1 μL T7 Enzyme Mix  1 μL Total Volume 10 μL

(1) 10× IVT Buffer—Tris/MgCl₂/DTT

TABLE 23 Component 1X Water 300 μL 1M Tris-HCl, pH 8 400 μL 1M DTT 100μL 1M MgCl₂ 200 μL Total Volume 1 ml

Recipes of salt and magnesium solutions:

TABLE 24   185 mM MgCl₂   10 μL 1M MgCl₂   44 μL Water   Total Volume 54 

TABLE 25   150 mM NaCl   3 μL 5M NaCl   97 μL Water   Total Volume 100μL  

(2) 10× IVT Buffer—Tris/MgCl₂/β-mercaptoethanol

TABLE 26 Component 1X Water 299 μL 1M Tris-HCl, Ph 8 400 μL 99% β-ME 101μL 1M MgCl₂ 200 μL Total Volume 1 ml

(3) 10× IVT Buffer—HEPES/MgCl₂/DTT

TABLE 27 Component 1X Water 300 μL 1M HEPES, pH 8 400 μL 1M DTT 100 μL1M MgCl₂ 200 μL Total Volume 1 ml

(4) 10× IVT Buffer—HEPES/MgCl₂/β-ME

TABLE 28 Component 1X Water 299 μL 1M HEPES, pH 8 400 μL 99% β-ME 101 μL1M MgCl₂ 200 μL Total Volume 1 ml

Reaction Scheme: All reactions used 1 μL of the Bulk Standard IAReaction described previously in table 20 in which the 10× EndonucleaseV buffer was 100 mM HEPES, pH=8, 15 mM MgCl₂, 0.1% tween-20, 2.5 mMdNTP, 10 mM DTT. This contained additional. MgCl₂ addition of 18.5 mM(20 mM [final]) and NaCl addition of 15 mM ([final]). The reactionmixtures used are shown in Table 29.

TABLE 29 ID Component 1 2 3 4 5 6 7 Water 3 1 3 2 3 2 3 rNTP Mix 4 4 4 44 4 4 10X IVT Buffer (Ambion) 1 1 — — — — — 10X Rxn Buffer A (describedabove) — — 1 1 — — — 10X Rxn Buffer B (described above) — — — — 1 1 —185 mM MgCl₂ — — — 1 — 1 — Glycerol — 2 — — — — — #1 10X IVT Buffer -Tris/MgCl₂/DTT — — — — — — 1 #2 10X IVT Buffer - Tris/MgCl₂/β-ME — — — —— — — Template 1 1 1 1 1 1 1 T7 Enzyme Mix (Ambion) 1 1 1 1 1 1 1 TotalVolume 10  10  10  10  10  10  10  ID Component 8 9 10 11 12 13 14 Water3 3 3 2 2 2 2 rNTP Mix 4 4 4 4 4 4 4 150 mM NaCl — — — 1 1 1 1 #1 10XIVT Buffer - Tris/MgCl₂/DTT — — — 1 — — — #2 10X IVT Buffer -Tris/MgCl₂/β-ME — — — — 1 — — #3 10X IVT Buffer - HEPES/MgCl₂/DTT — 1 —— — 1 — #4 10X IVT Buffer - HEPES/MgCl₂/β-ME — — 1 — — — 1 Template — 11 1 1 1 1 T7 Enzyme Mix 1 1 1 1 1 1 1 Total Volume 1 10  10  10  10  10 10 

Each reaction, 1-14, was incubated at 37° C. for 2 hours and stopped bythe addition of 20 μL GLB II. 3 μL/reaction were loaded into a well of a15% acrylamide 7M-urea TBE gel (Invitrogen). FIG. 6A shows thatping-pong amplification product is generated. The pong product thenbinds to primer P3 (the extender primer) and is extended with a promotersequence. FIG. 6B shows that the amplification product (ping-pongamplification product) is transcribed and corresponding RNA product (49nucleotide product) is made.

Example 6: Effect of SSB on DNA Amplification: Mixed Oligos; MgConcentration; and SSB Concentration

The following experiment demonstrates that the addition of more than 1ng of single strand binding protein to a 10 μL volume, increasesfidelity and reduces background amplification significantly.

TABLE 30 ID Component 1 2 3 4 5 6 7 8 9 10 11 12 13 14 DNAG5 1:10 − − −− − − − − + + + + + + DNAG5 1:100 − − − − − − − − − − − − − − 3 mMMgCl2 + + − − + + − − + + − − + + 6 mM MgCl2 − − + + − − + + − − + + − −12 Mix + + + + − − − − + + + + − − 3 Mix − − − − + + + + − − − − + + SSB1 ng/reaction + − + − + − + − + − + − + − SSB 1 μg/reaction − + − + − +− + − + − + − + ID Component 15 16 17 18 19 20 21 22 23 24 25 26 27 28DNAG5 1:10 + + − − − − − − − − + + + + DNAG5 1:100 −− + + + + + + + + + + − − 3 mM MgCl2 − − + + − − + + − − − − + + 6 mMMgCl2 + + − − + + − − + + + + − − 12 Mix − − + + + + − − − − − − + + 6Mix + + − − − − + + + + + − + − SSB 1 ng/reaction + − + − + − + − + −− + − + SSB 1 μg/reaction − + − + − + − + − +

Bulk Denaturation Mixes:

TABLE 31 ID Component A (X6) B (X6) C (X6) D (X6) E (X8) F (X8) 10X HEBuffer  6  6 6 6 8 8 DNAG5 1:10 — — 6 6 — — DNAG5 1:100 — — — — 8 8 12Mix  3 — 3 — 4 — 3 Mix —  3 — 3 — 4 Water 15 15 9 9 12  12  Total Volume24 24 24  24  32  32 

4 μL “A” were added to each of reactions 1-4; 4 μL of “B” were added toeach of reactions 5-8; 4 μL of “C” were added to each of reactions 9-12;4 μL of “D” were added to each of reactions 13-16; 4 μL “E” were addedto each of reactions 17-20, 25, 26; and 4 μL “F” were added to each ofreactions 21-24, 27, and 28.

TABLE 32 Bulk Enzyme Mixes: ID Component W (X9) X (X9) Y (X9) Z (X9) 10XReaction Buffer, 3 mM Mg⁺² 9 9 — — 10X Reaction Buffer, 6 mM Mg⁺² — — 99 100% Ethylene Glycol 9 9 9 9 E coli SSB, 1 ng/uL 9 — 9 — E coli SSB, 1μg/μL — 9 — 9 ΔTts (20 U/μl) 9 9 9 9 Endonuclease V (40 pmol/μL) 0.630.63 0.63 0.63 Water 17.37 17.37 17.37 17.37 Total Vol. 54 54 54 54 6 μLof “W” were added to each of reactions 1, 5, 9, 13, 17, 21, and 25; 6 μLof “X” were added to each of reactions 2, 6, 10, 14, 18, 22, and 26; 6μL of “Y” were added to each of reactions 3, 7, 11, 15, 19, 23, 27; and6 μL of “Z” were added to each of reactions 4, 8, 12, 16, 20, 24, and28.

Reactions 1-24 were incubated at 45° C. for 75 minutes and reactions25-28 were incubated 45° C. for 40 minutes. A 1/10th aliquot of eachreaction was analyzed on a 10% TB Urea gel (Invitrogen), which is shownin FIG. 7. In this example, the amount of template DNA, the number ofprimers, the divalent cation, and the amount of added SSB was varied todetermine if there was any effect on reaction sensitivity and off-targetprimer amplification products. The addition of 1 microgram of SSBreduced the artifactual primer amplification products in the absence ofadded DNA template (lanes 2, 4, 6 and 8). The use of 12 primers, 6 mMMgCl₂, and 1 microgram of SSB resulted in the most sensitive detectionof template DNA (lane 20), which was a product pattern than the6-primer, 6 mM MgCl₂, and 1 microgram of SSB (lane 24).

Example 7: Amplicon Extension Using Extension Templates: ExtenderTemplates; dd Terminators and Mismatched Primers

TABLE 33 12 Mix Dye-P-3, 4133-92 cP-3 3′ Quencher SEQ ID NO.: 23 P-3Mismatched SEQ ID NO.: 8 P-3SD Mod (ddC) SEQ ID NO.: 10 P-3 NO ddC SEQID NO.: 9 All oligos were diluted in TE + 0.01% Tween 20 to 10 pmol/uL

Reaction Scheme:

TABLE 34 ID Component 1 2 3 4 5 6 7 8 9 10 11 12 13 14 DNAG5 1:10 − + +− − − − + + + + + + + DNAG5 1:100 − − − + + − − − − − − − − − DNAG51:1000 − − − − − + + − − − − − − − 12 Mix + + + + + + + + + + + + − − 12Mix less SEQ ID NO.: 17 − − − − − − − − − − − − + + P-3 SD Mod − − − − −− − + − − − − − − P-3 NO ddC − − − − − − − − + − − − − − P-3 Mismatched− − − − − − − − − + − − − − cP-3 3′ Quencher − − − − − − − − − − + − − −Dye-P-3 − − − − − − − − − − − + − − ROX-11-ddG (3 pmol/) − − + − + − + −− − − − − +

Denaturations:

TABLE 35 ID Component 1 2 3 4 5 6 7 8 9 10 11 12 13 14 10X HE Buffer 1 11 1 1 1 1 1 1 1 1 1 1 1 DNAG1:10 — 1 1 — — — — 1 1 1 1 1 1 1 DNAG1:100 —— — 1 1 — — — — — — — — — DNAG1:1000 — — — — — 1 1 — — — — — — — 12 Mix0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 — — 12 Mix Less — — — —— — — — — — — — 0.5 0.5 SEQ ID NO.: 17 P-3SD Mod — — — — — — — 1 — — — —— — P-3N OddC — — — — — — — — 1 — — — — — P-3 — — — — — — — — — 1 — — —— Mismatched cP-33′ — — — — — — — — — — 1 — — — Quencher Dye-P-3 — — — —— — — — — — — 1 — — Water 2.5 1.5 1.5 1.5 1.5 1.5 1.5 0.5 0.5 0.5 0.50.5 1.5 1.5 Total Volume 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Bulk Enzyme Mix:

TABLE 36 Component 1X A (X12) B (X6) 10X Reaction Buffer 1 12 6 100%Ethylene Glycol 1 12 6 SYBR Green I (1:2000) 1 12 6 ROX-11-ddGTP 1 — 6ΔTts (20 U/uL) 1 12 6 Endonuclease V 0.075 0.9 0.45 SSB (1 ng/uL) 1 12 6Water — 12 — Total Vol. (uL) 6.075 72.9 36.45

6 μl of “A” were added to reaction mixtures 1, 2, 4, 6, and 8-13; and 6μl “B” were added to reaction mixtures 3, 5, 7, and 14. The resultantgel is depicted in FIG. 8. The result in lane 10 demonstrates that aprimer that can hybridize partially to the single stranded amplificationproducts of the reaction can support additional extension from the endsof those products generating a novel 3′ end, or potentially displacingpre-hybridized sequences. In lane 10, the additional primer used waspredicted to support the addition of 25 extra nucleotides onto the endof either the 88 nucleotide product, the 72 nucleotide product, or the45 nucleotide product. The arrows indicate the expected 113 nucleotide,97 nucleotide and 70 nucleotide extended products.

Example 8: Amplification Using Genomic DNA

Reaction Scheme:

TABLE 37 ID Component 1 2 3 4 5 6 7 8 9 10 11 DNAG5 1:10 − + + + + + − −− − − B. cereus gDNA (100 ng/uL) − − − − − − + + + + + SYBR Green I(1:2000) + + + + + + + + + + + SSB (100 ng/uL) − − + − − − − + − − − SSB(10 ng/uL) − − − + − − − − + − − SSB (1 ng/uL) − − − − + − − − − + − SSB(0.1 ng/uL) − − − − − + − − − − +

Denaturations:

TABLE 38 ID Component 1 2 3 4 5 6 7 8 9 10 11 10X HE Buffer 3 3 3 3 3 33 3 3 3 3 DNAG5 1:10 — 3 3 3 3 3 — — — — — B. cereus gDNA (100 — — — — —— 3 3 3 3 3 ng/uL) 4133-76OM n (Primer 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.51.5 1.5 1.5 Mixture of Table 34) Water 10.5 7.5 7.5 7.5 7.5 7.5 7.5 7.57.5 7.5 7.5 Total Volume (μL) 15 15 15 15 15 15 15 15 15 15 15

The reaction mixtures were heated 95° C. for 2 minutes and cooled toroom temperature in the thermal cycler. 3 μL of the indicated SSBsolution was added to the appropriate tubes

Bulk Enzyme Mix:

TABLE 39 Component 1X 13X (X3) 10X Rxn Buffer   1 μL    39 μL 1:2000SYBR Gold   1 μL    39 μL 100% Ethylene Glycol   1 μL    39 μL Exo (-)ΔTts   1 μL    39 μL Endo V 0.05 μL  1.95 μL Total Volume 4.05 μL 157.95μL

12 μL/reaction was cycled in an Applied Biosystems 7500 PCR System asfollows: (1) Stage 1, 10 seconds at 44° C.; and (2) Stage 2, 50 secondsat 45° C. Stages 1 and 2 were repeated seventy five times and data wascollected during stage 2. When completed, the reactions were stored at−20° C. overnight and then analyzed on a 10% TBE Urea gel. In thisreaction performed on both artificial DNA and genomic DNA, SSB effectwas being measured. The results indicate that the addition of SSB had adetrimental effect only on genomic DNA, and only at the highestconcentrations used as shown in FIG. 9.

Example 9: Effect of SSB on Amplification in the Presence ofContaminating DNA

The reaction mix contained 10 mM HEPES 7.9, 3 mM MgCl₂, 0.25 mM dNTP, 1mM DTT, 0.01% Tween, 10% ethylene glycol, 10% glycerol, 10 ng/μL E. coliSSB, 0.4 μM E. coli Endo V and 19 U delta Tts. Reaction mixturescontaining 0.5 ng of λ DNA, 5 ng λ DNA, 50 ng λ DNA, and the 6 primersshown in Table 40 (each primer at 1 μM concentration), were incubatedwith and without 100 ng of E. coli genomic DNA, and a titration of E.coli SSB was added as indicated in the chart at FIG. 10.

TABLE 40 6 primers Oligo used in the reactions: Length Ref. No.: 7290RAGTTCTTCTTTCGTCCCCIT 20 SEQ ID NO: 32 7270R CAGGCTGACATCACIIT 17SEQ ID NO: 33 7253R TCAGTTGTTCACCCAGCIA 19 SEQ ID NO: 34 7062FACCACCGGCGATCCIIC 17 SEQ ID NO: 38 7079F GCGTGAGTTCACCATIA 17SEQ ID NO: 39 7114F TGCTGCTGGCTGACCCTIA 19 SEQ ID NO: 53

Template and primers were pre-annealed by denaturation at 95° C. for 2min. then placed at room temperature, and then mixed with the rest ofreaction components. Reactions were incubated for 80 min. at 45° C. andproducts were separated on TBE-Urea gel. Gels were stained using SYBRgold and scanned on Typhoon scanner. As shown in FIG. 10, the SSBenhanced amplification of product (102 base pair products) in thepresence of contaminating genomic DNA.

Example 10: Generation and Expression of Y75A E. coli Endonuclease V andY74A Afu Endonuclease V

The original plasmid encodes wild type E. coli endo V in pET22b+ as aterminal HIS tagged fusion construct, flanked by an NdeI site at theinitiation and a XhoI site at the termination sequence. PCR wasperformed using primer “endo V us” with “endo V int ds” (SEQ ID NO: 26and SEQ ID NO: 29) to make a 215 base pair gene fragment. This fragmentwas restricted using NdeI and NgoI to generate cohesive ends. PCR wasperformed using primer “endo V ds” with “endo V int us” to make a 457base pair gene fragment. This fragment was restricted using NgoI andXhoI to generate cohesive ends. The fragments were ligated in a “3-way”reaction containing pET22b+ that had been linearized with NdeI and XhoI.After transformation into host E. coli strain JM109 (DE3), a clone wassequenced to confirm mutagenesis.

Protein was expressed and purified by standard techniques: TransformedE. coli JM109 (DE3) was grown in 2xYT medium and protein expressioninduced with IPTG. Protein was extracted into lysis buffer consisting of10 mM HEPES buffer (pH 8), 1 M NaCl, 0.1% TritonX-100, 0.1% Tween-20, 10mM β-ME, 5% glycerol, 10 mM Imidazole, with Roche “Complete” proteaseinhibitors. Sonication was used to disrupt the cells, and the extractclarified by centrifugation before application of the Ni-NTA resin.Captured protein was eluted into lysis buffer with 200 mM Imidazole.Batch mode capture, wash, and elution on Ni-NTA resin were preformedaccording to manufacturer recommendations (Qiagen Corp.). The eluate wasdialyzed and stored in a buffer consisting of 10 mM HEPES buffer (pH 8),150 mM NaCl, 0.1 mM EDTA, 0.01% TritonX-100, and 50% glycerol.

Y74A mutant Afu endonuclease V was prepared the same way as the E. colimutant, and has a sequence of SEQ ID NO: 3.

Example 12: Mutant Endo V Performance Characteristics

Reactions containing (25 mM Tris:borate (pH=8.1), 5 mM MgCl₂, 1 mM DTT,0.01% tween-20) and 1 pmole of FAM-I primer (SEQ ID NO: 24) and 1 pmoleHEX-C oligo (SEQ ID NO: 25), pre-annealed, were incubated at 37° C. for0, 5, 30, 60, 120, and 480 minutes with either 1, 0.3, or 0.1 pmoles ofwild type E. coli endo V or E. coli Y75A mutant endo V. Reactions wereresolved by denaturing acrylamide gel electrophoresis and gelsvisualized by scanning on a Typhoon 9410. The small molecular weightnicked product was quantified and the relative fluorescence was comparedand depicted in FIG. 11A and FIG. 11B. Results indicate that the mutantenzyme supports repeated nicking by each enzyme (when there is anInosine opposite to Cytosine), while the wild type enzyme seems capableof only a single round of nicking. The mutant enzyme supports repeatednicking by each enzyme (as evidenced by the extended kinetics; theability of enzyme to hop from one DNA to another), while the wild typeenzyme seems capable of only a single round of nicking (as evidenced bythe short burst kinetics).

Example 13: Inosine Specificity of E. coli Mutant Endo V

Reaction mixtures containing (25 mM Tris HCl (pH 8.5), 5 mM MgCl₂, 1 mMDTT, 0.01% tween20, 10% glycerol) were prepared with 200 ng of eitherHindIII linearized pUC18 DNA or 200 ng of HindIII restricted pUC18 DNAthat had been amplified using a modified rolling circle amplificationreaction in which the dGTP has been substantially replaced with dITP togenerate amplified material containing dIMP in place of dGMP, asindicated.

To the reaction was added either wild type or mutant E. coli endo V asindicated (0, 0.01, 0.1, or 1 microgram of protein), and incubated at37° C. for 90 minutes. Reactions were then visualized by denaturingagarose gel electrophoresis, staining with SYBR gold and scanning on theTyphoon 9410. Results shown in FIG. 12 indicate that both wild type andmutant enzyme have at least 100× increased nicking activity towardinosine-containing DNA as compared to gunanie-containing DNA.

Example 14: Amplification Reaction Using Mutant or Wild Type E. coliEndonuclease

Time course of amplification was studied in reaction mix containing 10mM Tris (pH 8.3), 2 mM MgCl₂, 0.01% Tween-20, 200 μM dNTP's, 10%Ethylene Glycol, 2 mM DTT, 25 ng/μL SSB, 10 nM exo (−) Bst polymerase,Large Fragment, and 10 nM E. coli Endo V Y75A mutant. DNA substrate was10 ng or 15 fmol of approximately 1 kb size PCR products made with I orG (penultimate base to 3′ end) containing PCR primers (described inTable 41). Reactions were stopped at 15, 30, 60, 120, 240, 480, and 1260min., by adding EDTA to 10 mM and samples were run on a 1% alkalineagarose gel to separate amplification products. Gels were thenneutralized and stained with SYBR gold and scanned and quantified onTyphoon 9410 and ImageQuant image analysis software, as shown in FIG.13.

TABLE 41 Name Sequence Length Ref. No. : Forward primerGTTTTCCCAGTCACGACGTTGTA 34 SEQ ID NO: 47 40FGI AAACGACGICCReverse primer: TCAAAGAAGTATTGCTACAACG 23 SEQ ID NO: 48 GForward primer: GTTTTCCCAGTCACGACGTTGTA 34 SEQ ID NO: 49 40FGGAAACGACGGCC Reverse primer: TCAAAGAAGTATTGCTACAACG 23 SEQ ID NO: 50 G

Example 15: Kinetics of Amplification: Comparison of E. coli Y75A MutantEndo V Versus Afu. Y74A Mutant Endo V

TABLE 42 Name Sequence Length Ref. No.: Tban-1 DNA CAATAGACTCCATACCACCAA112 SEQ ID NO: 54 Template TTGTAATTTCTGTACGTCTCTT ATCATTGAAGCGCTCTTTTACTTCTGTTAATTCTTCACGAATAA TCTCAAGAACCTTTTCTTCATC TGC Tban-1 forwardGCAGATGAAGAAAAGGTTCTT  33 SEQ ID NO: 51 primer: GAGATTATTCITTban-1 reverse CAATAGACTCCATACC  34 SEQ ID NO: 55 primerACCAATTGTAATTTCTIT

Amplification reactions were carried out in reaction mix containing 10mM Tris (pH 8.3), 3 mM MgCl₂, 0.01% Tween-20, 250 μM dNTP's, 10%Ethylene Glycol, 1 mM DTT, 50 nM exo (−) Bst polymerase large Fragment,or Tma polymerase (100 ng), and 0.8 μM E. coli Endo V, E. coli Y75Amutant endo V or 0.4 μM Afu, Endo V, Afu Y75A mutant endo V. Bothforward and reverse primers (described in Table 42) were maintained at0.25 μM and template at 0.1 μM.

Reactions were incubated for 80 min. at 45° C. and products wereseparated on TBE-Urea gel. Gels were stained using SYBR gold and scannedon Typhoon scanner as shown on FIG. 14. In both cases the correctamplification products of 45 nucleotides and 79 nucleotides wereobserved.

Example 16: Thermal Stability of Mutant Endo V

E. coli Y75A mutant Endo V at a concentration of 1.6 μM was incubated atfollowing temperatures: on ice, 37° C., 40° C., 43° C., 46° C., 49° C.,52° C., 55° C., and 58° C. for different amount of times such as 15, 30,45, 60, and 75 min., in buffer containing 10 mM HEPES (pH 7.9), 3 mMMgCl₂, 0.25 mM dNTP, 1 mM DTT, 0.01% Tween, 10% ethylene glycol, and 0.5μL ThermoFidelase (Fidelity Systems).

At time points indicated above samples were removed from the incubatorand put on ice until all incubations were completed. To thesepre-incubated samples following reaction components were added (10 mMHEPES (pH 7.9), 0.25 mM dNTP, 1 mM DTT, 0.01% Tween, 10% ethyleneglycol) and P1/P2-1/P3 primer mix (to 0.2/1/1.8 μM each), 0.5 nMtemplate, 10 ng/μL of T4 g32 protein, 5U exo(−) Klenow polymerase.

All reactions were then incubated at 45° C. for 80 min. Products wereseparated on TBE-Urea gel that was stained using SYBR gold and scannedon Typhoon scanner. The results are shown in FIG. 15. The endonuclease Vmutant maintains stability up to 46° C. for 75 minutes, and wasinactivated at 49° C.

Example 17: Real Time Analysis of Amplicon Generation

A dilution series of lambda DNA was prepared in HETB buffer (10 mM HEPES(pH 7.9), 0.1 mM EDTA, 0.01% tween-20, 0.5 mg/ml BSA) and 10 pmoles eachof the following primers: (7062F, 7079F, 7096F, 7111F, 7127F, 7144F,7290R, 7270R, 7253R, 7234R, 7215R, 7194R, shown below in Table 43) andheat denatured for 2 minutes at 95° C. and then chilled on ice.

TABLE 43 Oligo Length 7290R 20 7270R 17 7253R 19 7234R 19 7215R 17 7194R19 7062F 17 7079F 17 7096F 18 7111F 16 7127F 17 7144F 15 T7-7158F-misA:50 Quencher-oligo 24 Dye-oligo 24

A mix containing the following components was then added to a finalconcentration of: 35 units delta Tts, 8 pmoles E. coli Y75A endo Vmutant, 0.002 mg SSB, 3 mM MgCl₂, 0.1 mM MnSO4, 3 pmole ROX std dye(internal control), 1× buffer (20 mM HEPES (pH 7.9), 0.25 mM dNTP, 1 mMDTT, 0.01% Tween, 10% glycerol, and 10% ethylene glycol. Then 3 pmolesT7-7158F-misA, 4 pmoles quencher-oligo, 2.5 pmoles dye-oligo were mixedin HET buffer, heated to 95° C. for 45 seconds and slow cooled to iceover 5 minutes and added to the amplification reaction.

The reactions were cycled an ABI 7500 75 times (46° C., 50 seconds; 45°C., 10 second), taking readings during the 46° C. step. This 1° C. cyclewas employed because the ABI machine does not hold reactions at a singletemperature. A 75-minute incubation was performed and data was exportedto excel. The time at which each reaction produced a signal above athreshold level was plotted on a semi log scale. A linear curve wasgenerated (shown in FIG. 16, amplification time v. initial DNAconcentration), indicating that the reaction gave reliablequantification over at least 5 orders of magnitude.

Example 18: Nicking Efficiency of Y80A Mutant Tma Endonuclease V (SEQ IDNO: 58) on Double Stranded DNA and Single Stranded DNA

The indicated amount of either wild type Tma endoV or Y80A mutant TmaendoV was incubated with 3 pmoles of fluorescent primer 1 (SEQ ID NO:24) alone or annealed to fluorescent primer 2 (SEQ ID NO: 25), in 1×reaction buffer at 60 degrees for 30 minutes. Reactions were separatedon denaturing polyacrylamide and visualized by scanning for fluoresceinfluorescence. The amount of nicked product was quantified and reportedin the graph. Nicking efficiency of Y80A mutant Tma endonuclease V (SEQID NO: 58) on double stranded DNA and single stranded DNA is shown FIG.17. It was observed that the mutant Tma endonuclease V nicks a singlestranded DNA faster than a double stranded DNA.

Example 19: Nicking of Double Stranded DNA and Single Stranded DNA byTma Endonuclease V at 45 C and 60 C

The indicated amount of either wild type Tma endo V or Y80A mutant Tmaendo V was incubated with 3 pmoles of fluorescent primer 1 (SEQ ID NO:24, referred in FIG. 18 as “F”) alone or annealed to fluorescent primer2 (SEQ ID NO: 25, referred in FIG. 18 as “H”), in 1× reaction buffer ateither 45 or 60 degrees, as indicated, for 30 minutes. Reactions wereseparated on denaturing polyacrylamide and visualized by scanning forfluorescein fluorescence.

FIG. 18 shows that the Y80A mutant Tma endo V is more active at 60degrees than at 45 degrees, and this may indicate that this highertemperature is required for the enzyme to dissociate from a nickedproduct effectively.

Example 20: Nicking of Double Stranded DNA Y80A Tma Endonuclease V at 60C in the Presence of Various Additives

4 ng of Y80A mutant Tma endo V was incubated 15 minutes at 60 degrees inreaction buffer (25 mM HEPES (pH=8), 3 mM MgCl₂, 0.1 mM MnSO₄, 250 μMeach dNTP (all 4), 1 mM TCEP, 0.1 mM EDTA, 0.02% Tween 20, 5 mM(NH4)₂SO₄), containing 3 pmoles of fluorescent primer 1 (SEQ ID NO: 24,referred in FIG. 19 as “FAM”) annealed to fluorescent primer 2 (SEQ IDNO: 25, referred in FIG. 19 as “HEX”) and the resulting denaturingpolyacrylamide gel scanned for fluorescein (FAM) and forhexachloro-fluorescein (HEX).

FIG. 19 shows that the strand containing inosine is the only that getsnicked under any condition. Also, that the addition of 1 μg of E. coliSSB inhibits Y80A mutant Tma endo V nicking, and that this inhibition isonly slightly relieved by the addition of 20% ethylene glycol and 10%glycerol. This is quite different from what is observed with Y75A E.coli endo V in which the addition of SSB is not inhibitory at all. IfSSB is required for effective strand displacement synthesis in a DNAamplification such as one described herein, the Y75A E. coli endo V maybe preferred over the Y80A Tma endo V.

Example 21: Nicking Efficiency of Various Mutant Endonuclease V Enzymeson Double Stranded DNA and Single Stranded DNA

Nicking efficiency of either Y75A mutant E. coli endonuclease V (SEQ IDNO: 2) or Y80A mutant Tma endonuclease V were tested on 3 pmoles offluorescent primer 1 (SEQ ID NO: 24) alone or annealed to 3 pmoles offluorescent primer 2 (SEQ ID NO: 25), as indicated.

The nicking reaction is performed under the following conditions: 25 mMHEPES (pH=8), 3 mM MgCl₂, 0.1 mM MnSO₄, 250 μM each dNTP (all 4), 1 mMTCEP, 0.1 mM EDTA, 0.02% Tween 20, 5 mM (NH₄)₂SO₄. Reactions wereincubated either at 45 degrees or 60 degrees as indicated and resolvedon denaturing polyacrylamide and visualized by scanning for fluorescein.

FIG. 20 demonstrate that the Y75A mutant E. coli endonuclease V (SEQ IDNO: 2) is more effective in nicking a double stranded DNA than a singlestranded DNA as compared to Y80A mutant Tma endonuclease V (SEQ ID NO:58, wherein a Tyrosine residue at the 80^(th) position of a WT Tma endoV (SEQ ID No: 57) is replaced with an Alanine residue), which is moreeffective at nicking single stranded DNA than double stranded DNA at 45°C., but about equally active towards both substrates at 60° C. Thissuggests that if amplification of DNA by the methods described hereinare to be attempted, the Y80A mutant Tma endo V may require incubationat 60° C. or higher, while the Y75A E. coli endo V can be used at 45° C.

Example 22: Nicking Efficiency of Y75A Mutant E. coli Endonuclease V(SEQ ID NO: 2) on Single Stranded DNA and Double Stranded DNA in VariousBuffers

The nicking experiments were performed on 3 pmoles of fluorescent primer1 (SEQ ID NO:24) annealed to fluorescent primer 2 (SEQ ID NO: 25) in thefollowing buffers. Buffer (1): 10 mM HEPES (pH=8.0), 3 mM MgCl₂, 0.01%Tween 20 and 1 mM TCEP; Buffer (2): Buffer (1)+0.1 mM MnSO₄; Buffer (3):Buffer (1)+250 μM dATP; Buffer (4): Buffer (1)+250 μM dCTP; Buffer (5):Buffer (1)+250 μM dGTP; Buffer (6): Buffer (1)+250 μM dTTP; Buffer (7):Buffer (2)+250 μM each of dNTP (all 4); Buffer (8): 10 mM TRIS(pH=8.0)+2 mM MgCl₂, 0.01% Tween 20 and 1 mM TCEP.

FIG. 21 demonstrates that Y75A mutant E. coli endonuclease V nickssingle stranded DNA at a slower rate than the double stranded DNA.Addition of Mn²⁺ increases activity on single stranded DNA as comparedto that of double stranded DNA. However, addition of any dNTP to thesingle stranded DNA nicking reaction with the presence of Mn²⁺ the endoactivity on single stranded DNA to the rate seen with single strandedDNA without Mn²⁺.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are selected embodiments or examples from a manifold of allpossible embodiments or examples. The foregoing embodiments aretherefore to be considered in all respects as illustrative rather thanlimiting on the invention described herein. While only certain featuresof the invention have been illustrated and described herein, it is to beunderstood that one skilled in the art, given the benefit of thisdisclosure, will be able to identify, select, optimize or modifysuitable conditions/parameters for using the methods in accordance withthe principles of the present invention, suitable for these and othertypes of applications. The precise use, choice of reagents, choice ofvariables such as concentration, volume, incubation time, incubationtemperature, and the like may depend in large part on the particularapplication for which it is intended. It is, therefore, to be understoodthat the appended claims are intended to cover all modifications andchanges that fall within the true spirit of the present invention.Further, all changes that come within the meaning and range ofequivalency of the claims are intended to be embraced therein.

The invention claimed is:
 1. A nucleic acid assay, comprising: providinga double stranded DNA that contains an inosine residue base-paired witha cytosine residue; nicking the inosine-containing strand of the doublestranded DNA at a residue 3′ to the inosine residue using one or more ofa mutant endonuclease V comprising an amino acid sequence of SEQ ID NO:1, wherein its Tyrosine 75 residue is replaced with Alanine, a mutantendonuclease V comprising an amino acid sequence of SEQ ID NO: 2, or amutant Archaeoglobus fulgidus endonuclease V comprising an amino acidsequence of SEQ ID NO: 3; and performing the nucleic acid assay.
 2. Thenucleic acid assay of claim 1, wherein the double stranded DNA isgenerated in situ by a primer-mediated replication of a target DNA. 3.The nucleic acid assay of claim 2, comprising extending the nickedinosine-containing strand via a nucleic acid amplification reactionusing a strand displacement DNA polymerase.
 4. The nucleic acid assay ofclaim 3, wherein the nucleic acid amplification is an isothermalamplification.
 5. A nucleic acid assay comprising: providing a targetDNA; providing a DNA polymerase; providing a mutant endonuclease Vcomprising an amino acid sequence of SEQ ID NO: 2; generating a doublestranded DNA from the target DNA, wherein the double stranded DNAcomprises an inosine residue base-paired with a cytosine residue;nicking the inosine-containing strand of the double stranded employingthe mutant endonuclease V to generate a nicked DNA; and performing a DNApolymerase reaction on the nicked DNA employing the DNA polymerase.
 6. Anucleic acid assay comprising: providing a target DNA; providing a DNApolymerase; providing a mutant endonuclease V comprising an amino acidsequence of SEQ ID NO: 1, wherein its Tyrosine 75 residue is replacedwith Alanine; generating a double stranded DNA from the target DNA,wherein the double stranded DNA comprises an inosine residue base-pairedwith a cytosine residue; nicking the inosine-containing strand of thedouble stranded employing the mutant endonuclease V to generate a nickedDNA; and performing a DNA polymerase reaction on the nicked DNAemploying the DNA polymerase.
 7. A nucleic acid assay comprising:providing a target DNA; providing a DNA polymerase; providing a mutantArchaeoglobus fulgidus endonuclease V comprising an amino acid sequenceof SEQ ID NO: 3; generating a double stranded DNA from the target DNA,wherein the double stranded DNA comprises an inosine residue base-pairedwith a cytosine residue; nicking the inosine-containing strand of thedouble stranded employing the mutant endonuclease V to generate a nickedDNA; and performing a DNA polymerase reaction on the nicked DNAemploying the DNA polymerase.